Active-site engineering of nucleotidylyltransferases and general enzymatic methods for the synthesis of natural and &#34;unnatural&#34; UDP- and TDP-nucleotide sugars

ABSTRACT

The present invention provides mutant nucleotidylyl-transferases, such as E p , having altered substrate specificity; methods for their production; and methods of producing nucleotide sugars, which utilize these nucleotidylyl-transferases. The present invention also provides methods of synthesizing desired nucleotide sugars using natural and/or modified Ep or other nucleotidyltransferases; and nucleotide sugars sythesized by the present methods. The present invention further provides new glycosyl phosphates, and methods for making them.

[0001] This application claims the benefit of U.S. application Ser. No.60/254,927, filed Dec. 13, 2000.

FIELD OF THE INVENTION

[0002] The present invention is directed to nucleotidylyl-transferasesand mutant nucleotidylyltransferases having altered substratespecificity and methods for their production.

[0003] The present invention is also directed to methods of synthesizingdesired nucleotide sugars using natural and/or mutant E_(p) or othernucleotidyltransferases, preferably E_(p) or othernucleotidylyltransferases modified by the present methods. Additionally,the present invention is directed to nucleotide sugars sythesized by thepresent methods.

[0004] The present invention is further directed to new glycosylphosphates, and methods for making them.

BACKGROUND

[0005] Many bioactive metabolites possess unusual carbohydrates requiredfor molecular recognition. (See for example, Liu, H.-w.; Thorson, J. S.Ann. Rev. Microbiol., 1994, 48, 223-256; Weymouth-Wilson, A. C. Nat.Prod. Rep. 1997, 14, 99-110; In Macrolide Antibiotics, Chemistry,Biology and Practice; Omura, S. Ed., Academic Press: New York; 1984;Johnson, D. A.; Liu, H.-w. Curr. Opin. Chem. Biol. 1998, 2, 642-649; andTrefzer, A.; Salas, J. A.; Bechthold, A. Nat. Prod. Rep. 1999, 16,283-299.) In fact, roughly 70% of current lead compounds in modern drugdiscovery derive directly from natural products, many of which areglycosylated metabolites. (See Thorson, J. S. et al. Nature'sCarbohydrate Chemists: The Enzymatic Glycosylation of BioactiveBacterial Metabolites. Curr. Org. Chem. manuscript in press, (2000); andreferences therein and Weymouth-Wilson, A. C. The Role of Carbohydratesin Biologically Active Natural Products. Nat. Prod. Rep. 14, 99-110(1997)). Examples of pharmaceutically important glycosylated metabolitesinclude, for example, amphotericin, megalomicin/erythromycin,mithramycin, doxorubicin, vancomycin and calicheamicin, as shown in FIG.5. While it is known that the sugar moieties of these pharmaceuticallyimportant metabolites often define their corresponding biologicalactivity, (see Weymouth-Wilson, A. C., The Role of Carbohydrates inBiologically Active Natural Products, Nat. Prod. Rep. 14, 99-110(1997)), efficient methods to systematically alter these essentialcarbohydrate ligands are still lacking.

[0006] In metabolite biosynthesis, glycosylation begins with thenucleotidylyltransferase-catalyzed activation of a sugar phosphate as anucleotide diphosphosugar (NDP-sugar) donor. After activation, a numberof enzymatic processing reactions often occur (e.g., deoxygenation,transamination, oxidation/reduction, epimerization, alkylation, anddecarboxylation) prior to the culminating glycosyltransferase-catalyzedattachment to the aglycon. (Liu, H.-w. & Thorson, J. S. Pathways andMechanisms in the Biogenesis of Novel Deoxysugars by Bacteria. Ann. Rev.Microbiol. 48, 223-256 (1994); Kirschning, A., Bechtold, A. F-W. & Rohr,J. Chemical and Biochemical Aspects of Deoxysugars and DeoxysugarOligosaccharides. Top. Curr. Chem. 188, 1-84 (1997); Johnson, D. A. &Liu, H.-w. Mechanisms and Pathways from Recent Deoxysugar BiosynthesisResearch. Curr. Opin. Chem. Biol. 2, 642-649 (1998); Hallis, T. M. &Liu, H.-w. Learning Nature's Strategies for Making Deoxy Sugars:Pathways, Mechanisms, and Combinatorial Applications. Acc. Chem. Res.32, 579-588 (1999); Johnson, D. A. & Liu, H.-w. In ComprehensiveChemistry of Natural Product Chemistry (Barton, D.; Nakanishi; K.;Meth-Cohn, O. eds), Elsevier Science, Oxford, 311, (1999); Trefzer, A.,Salas, J. & Bechthold, A. Genes and Enzymes Involved in DeoxysugarBiosynthesis in Bacteria. Nat. Prod. Rep. 16, 283-299 (1999); andBechthold, A. & Rohr, J. In New Aspects of Bioorganic Chemistry(Diederichsen, U.; Lindhorst, T. K.; Wessjohann, L.; Westerman, B.,eds.) Wiley-VCH, Weinheim, 313, (1999)).

[0007] The glycosyltransferases that incorporate these essential ligandsare thought to rely almost exclusively upon UDP- and TDP-nucleotidesugars; however some have demonstrated promiscuity towards the sugardonor, (e.g., Gal, D-galactose; Glc, D-glucose; Man, D-mannose; NTP,nucleotide triphosphate; pFPTC, pentafluorophenoxythiocarbonyl; TDP,thymidine diphosphate; TMP, thymidine monophosphate; TTP, thymidinetriphosphate; UDP, uridine diphosphate.) Genetic experiments suggestthat downstream glycosyltransferases in secondary metabolism arepromiscuous with respect to their NDP-sugar donor, setting the stage forthe expansion of “combinatorial biosynthesis” approaches to changemetabolite glycosylation. (See Madduri, K. et al., Production of theantitumor drug epirubicin (4′-epidoxorubicin) and its precursor by agenetically engineered strain of Streptomyces peucetius Nat. Biotech.16, 69-74 (1998); and Hutchinson, C. R. Combinatorial Biosynthesis forNew Drug Discovery. Curr. Opin. Microbiol. 1, 319-329 (1998).) Thisinformation has led to the exploitation of the carbohydrate biosyntheticmachinery to manipulate metabolite glycosylation, (Madduri, K.; Kennedy,J.; Rivola, G.; Inventi-Solari, A.; Filppini, S.; Sanuso, G.; Colombo,A. L.; Gewain, K. M.; Occi, J. L.; MacNeil, D. J.; Hutchinson, C. R.Nature Biotech. 1998, 16, 69-74; and Zhao, L.; Ahlert, J.; Xue, Y.;Thorson, J. S.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc., 1999, 121,9881-9882 and references therein), revitalizing interest in methods toexpand the repertoire of available UDP- and TDP-sugar nucleotides. (SeeZhao, Y.; Thorson, J. S. J. Org. Chem. 1998, 63, 7568-7572; andElhalabi, J. M.; Rice, K. G. Cur. Med. Chem. 1999, 6, 93-116.)

[0008] These in vivo methods are limited by both a particular host'sbiosynthetic machinery and the specific host's tolerance to each newlyconstructed metabolite. Further, in vitro progress in this area islimited by the availability of the required NDP-sugar substrates.(Solenberg, P. J. et al., Production of Hybrid Glycopeptide Antibioticsin vitro and in Streptomyces toyocaensis. Chem. & Biol. 4, 195-202(1997).)

[0009] Thus, there is a need for a greater variety of availableNDP-sugar substrates.

[0010]Salmonella enterica LT2 α-D-glucopyranosyl phosphatethymidylyltransferase (E_(p)) is a member of the prevalentnucleotidylyltransferase family responsible for the reversibleconversion of α-D-hexopyranosyl phosphate and NTP to the correspondingNDP-sugar nucleotide and pyrophosphate. Of the manynucleotidylyl-transferases studied, the NDP-sugar nucleotide-formingthymidylyltransferases have received the least attention in prior work.(See Lindquist, L.; Kaiser, R.; Reeves, P. R.; Lindberg, A. A. Eur. J.Biochem. 1993, 211, 763-770, and Gallo, M. A.; Ward J.; Hutchinson, C.R. Microbiol. 1996, 142, 269-275.) Even in E_(p), substrate specificitystudies prior to the work of the present inventors were limited to onlya few available hexopyranosyl phosphates. (See Lindquist, L.; Kaiser,R.; Reeves, P. R.; Lindberg, A. A. Eur. J. Biochem. 1993, 211, 763-770.)

SUMMARY OF THE INVENTION

[0011] The present invention is directed to methods of engineering ormutating nucleotidylyltransferases, such as E_(p), to vary theirspecificity in a directed manner. The invention is also directed tonucleotidylyl-transferases and mutated nucleotidyltransferases,preferably E_(p) or other nucleotidyltransferases modified by thepresent methods. The present invention is further directed to mutantE_(p) and other nucleotidyltransferases with altered substratespecificity, methods for their production, and methods of producingnucleotide sugars, which utilize these nucleotidyl-transferases.

[0012] The present invention is also directed to methods of synthesizingdesired nucleotide sugars using natural and/or mutated E_(p) or othernucleotidylyltransferases, preferably E_(p) or othernucleotidylyltransferases mutated by the present methods. Additionally,the present invention is directed to nucleotide sugars sythesized by thepresent methods.

[0013] Examples of nucleotide sugars produced the present methods (thatis, via the exploitation of the promiscuity of E_(p)) include, but arenot limited to Thymidine 5′-(α-D-glucopyranosyl diphosphate) (58);Uridine 5′-(α-D-glucopyranosyl diphosphate) (59); Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60); Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61); Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62); Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63); Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64); Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65); Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66); Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67); Thymidine5′-(α-D-mannopyranosyl diphosphate) (68); Uridine 5′-(α-D-mannopyranosyldiphosphate) (69); Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);Uridine 5′-(α-D-galactopyranosyl diphosphate) (71); Thymidine5′-(α-D-allopyranosyl diphosphate) (72); Uridine 5′-(α-D-allopyranosyldiphosphate) (73); Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);Uridine 5′-(α-D-altropyranosyl diphosphate) (75); Thymidine5′-(α-D-gulopyranosyl diphosphate) (76); Uridine 5′-(α-D-gulopyranosyldiphosphate) (77); Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);Uridine 5′-(α-D-idopyranosyl diphosphate) (79); Thymidine5′-(α-D-talopyranosyl diphosphate) (80); Uridine 5′-(α-D-talopyranosyldiphosphate) (81); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (109); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (110); Thymidine 5′-(4-amino-4-deoxy-α-D-lucopyranosyldiphosphate) (111); Uridine 5′ (4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (112); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (113); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (114); Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (115); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (116); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (117); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (118); Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (119); Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (120); Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (121); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (122); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (123); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (124); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (125); and Uridine5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (126).Nucleotide sugars such as these, and methods for making them, areprovided by the present invention.

[0014] Examples of nucleotide sugars according to the present invention,which may be produced by designed mutants of E_(p) include, but are notlimited to, Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (117); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (118); Thymidine 5′-(α-D-glucopyran-6-uronic aciddiphosphate) (130); Uridine 5′-(α-D-glucopyran-6-uronic aciddiphosphate) (131); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (123); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (124); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (125); Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (126); Thymidine 5′-(α-D-arabinopyranosyl diphosphate)(128); and Uridine 5′-(α-D-arabinopyranosyl diphosphate) (129). Thesenucleotide sugars, and methods for making them, are provided by thepresent invention.

[0015] The present invention is also directed to new glycosylphosphates, and methods for making them. Examples of these new glycosylphosphates and methods for synthesizing them are represented for examplein FIG. 1(b).

[0016] The present inventors have discovered that E_(p) is pliable interms of its substrate specificity. The present inventors have alsodiscovered the three dimensional structure of E_(p) and the moleculardetails of E_(p) substrate recognition.

[0017] In general, the present invention provides a very rapid method ofconverting sugar phosphates to nucleotide diphosphosugars.

[0018] The present invention will broadly impact efforts to understandand exploit the biosynthesis of glycosylated bioactive natural products,many of which are pharmacologically useful. (See Thorson, J. S.; Shen,B.; Whitwam, R. E.; Liu, W.; Li, Y.; Ahlert, J. Bioorg. Chem., 1999, 27,172-188; Whitwam, R. E.; Ahlert, J.; Holman, T. R.; Ruppen, M.; Thorson,J. S. J. Am. Chem. Soc., 2000, 122, 1556-1557; Thorson, J. S.; Sievers,E. L.; Ahlert, J.; Shepard, E.; Whitwam, R. E.; Onwueme, K. C.; Ruppen,M. Cur. Pharm. Des., 2000, manuscript in press; and J. S. Thorson, T. J.Hosted Jr., J. Jiang, J. B. Biggins, J. Ahlert, M. Ruppen, Curr. Org.Chem. 2000.)

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1. FIG. 1(a) depicts a reaction according to the presentinvention, catalyzed by E_(p). In this reaction, the enzyme catalyzesthe reversible conversion of an α-D-hexopyranosyl phosphate (such as anα-D-glucopyranosyl phosphate)(2) and NTP, such as TTP (1) to thecorresponding NDP-sugar nucleotide (for example a TDP-sugar nucleotide,such as TDP-Glc)(3) and pyrophosphate (4). Glc1P (2) depicted in thereaction of FIG. 1(a) is a commercially available α-D-hexopyranosylphosphate (although other α-D-hexopyranosyl phosphates that may be usedin accordance with the present invention may include those synthesizedfrom free sugars.)

[0020]FIG. 1(b) depicts the synthesis of α-D-hexopyranosyl phosphates.

[0021]FIG. 2. E_(p)-Catalyzed Conversion of Substrates (a) Percentconversion=[A_(P)/(A_(P)+A_(T))]×100, where A_(P) is the NDP-sugarproduct peak integration and A_(T) represents the NTP peak integration.HRMS for all observed products reported in the supporting information.(b) Standard retention times: TDP, 4.5 min; TTP, 7.2 min; UDP, 4.0 min;UTP, 6.1 min. (c) Commercially available. (d) Coelutes with commerciallyavailable standard. (e) Product hydrolysis observed (43, 7.6% TDP and10.2% UDP). (f) Adjusted for the 2:1 α/β-28. (g) In contrast topreviously published studies (See Lindquist, L; Kaiser, R.; Reeves, P.R.; Lindberg, A. A., Eur J. Biochem, 1993, 211, 763-770). (h) Noproducts observed.

[0022]FIG. 3. FIG. 3(a) sets forth a reaction according to the presentinvention, catalyzed by E_(p). FIG. 3(b) shows an overview of the keysteps in the described syntheses of E_(p) substrates analogs. The boxhighlights the point from which the aminodeoxy-α-D-glucose phosphateseries and N-acetyl-aminodeoxy-α-D-glucose phosphate series diverge. Thereaction conditions of the steps are as follows: (a) TMSSEt, ZnI₂ (84.2%overall yield); (b) i) MeONa, ii) NaH, BnBr (77.3% average overallyield, two steps) (c) i) SnCl₂, PhSH, Et₃N, ii) Ac₂O, pyr (84.0% averageoverall yield, two steps); (d) i) Tf₂O, pyr, ii) NaN₃ (87.7% averageoverall yield, two steps); (e) i) NaOMe, ii) CH₃CH(OCH₃)₂CH₃, TsOH, iii)NaH, BnBr, iv) HCl/MeOH, v) BzCl, DMAP, Et₃N (87.3% average overallyield, five steps); final steps (not shown): i) phosphorylation, ii)reductive deprotection, iii) cation exchange to give the Na⁺ salt (44.4%average overall yield).

[0023]FIG. 4. Percent conversion to product using substrates accordingto the present invention.

[0024]FIG. 5. Examples of pharmacologically important glycosylatedmetabolites. The general nucleotidylyl-transferase-catalyzed formationof NDP-sugars is highlighted in the box while the carbohydrate ligandsof each metabolite are accentuated in red. Note the difference betweenerythromycin from S. erythrea and megalomicin from M. megalomicea is theaddition of a third sugar megosamine (highlighted by the arrow).

[0025]FIG. 6. Representative region of the density-modified experimentalelectron density map showing the substrate binding pocket in the E_(p)UDP-Glc structure configured at 1.2 σ.

[0026]FIG. 7 Quaternary structure of E_(p) bound to UDP-Glc or dTTP. (a)Two 90 degree views of the E_(p) tetramer bound to four molecules ofUDP-Glc. (b) The E_(p) tetramer bound to eight molecules of dTTP.

[0027]FIG. 8. Structures of the E_(p) monomer and structural homologsSpsA and GlmU. The β strands and α helices of the α/β open sheetRossmann fold are shown in red and green respectively, while variableregions are shown in yellow. (a) Two 90 degree views of the E_(p)monomer (upper) and the corresponding structures of of SpsA (lower left)and GlmU (lower right). (b) The folding topology of E_(p), SpsA, andGlmU.

[0028]FIG. 9. Close up views of the E_(p) active site. Hydrogen bondsare depicted by green dashed lines. (a) Interactions between E_(p) andthe dTTP substrate (left) and the UDP-Glc product (right). (b)Interactions between E_(p) and the glucose moiety in the sugar bindingpocket. (c) Two different views of dTTP bound in the ‘accessory’ site atthe monomer interface. The different chains of the tetramer are labeledeither in blue (chain-A) or red (chain-B). The β-phosphate of dTTPhydrogen bonds with both His117 of chain-A and Gly221 of chain-B.

[0029]FIG. 10. (a) The proposed enzymatic mechanism based on thestructures of substrate- and product-bound E_(p). (b) The determinationof E_(p) steady state kinetic parameters. The conditions for the E_(p)assay conditions and HPLC resolution of reactants and products weresimilar to those described in Jiang, J., Biggins, J. B. & Thorson, J. S.A General Enzymatic Method for the Synthesis of Natural and “Unnatural”UDP- and TDP-Nucleotide Sugars. J. Am. Chem. Soc. 122, 6803-6804 (2000).The Lineweaver-Burke plots of from assays (done in triplicate) varyingdTTP concentration as a function of α-D-glucose-1 -phosphateconcentration (mM): 0.5 (□), 0.3 nM (o), 0.2 (⋄), 0.1 (Δ) and 0.05 (▪).(c) Secondary plot from FIG. 6b (dTTP K_(m)=0.7±0.2; V_(max)=0.03±0.01mM min⁻¹). (d) The Lineweaver-Burke plots of assays (done in triplicate)varying α-D-glucose-1-phosphate concentration as a function of dTTPconcentration (mM): 0.25 (□), 0.15 nM (o), 0.1 (⋄), 0.05 (Δ) and 0.02(▪). (e) Secondary plot from FIG. 6d (α-D-glucose-1-phosphateK_(m)=0.3±0.1; V_(max)=0.03 ±0.02 mM min⁻¹).

[0030]FIG. 11. Percent conversion of sugar phosphates according to thepresent invention by wild-type and mutant enzymes. The alterations fromnative substrate (Glc-1-P,1) are highlighted in red. For the mutantpool, mutants Asp41Asn, Glu62Asp, Thr201A and Trp224His were pooled,concentrated and an aliquote constituting 60 μg of each mutant(corresponding to 3.5U E_(p)) was utilized for the assay.

[0031] Percent conversion was determined as described in Jiang, J.,Biggins, J. B. & Thorson, J. S. A General Enzymatic Method for theSynthesis of Natural and “Unnatural” UDP- and TDP-Nucleotide Sugars. J.Am. Chem. Soc. 122, 6803-6804 (2000).

[0032] §Represents less than 5% conversion to product.

[0033]FIG. 12. Shows alignment of the Thymidylyltransferase sequence.

[0034]FIG. 13. Shows alignment of the E_(p) sequence with Glc-1-PUridylyltransferases.

[0035]FIG. 14. Shows alignment of the E_(p) sequence with Glc-1-PAdenylyltransferases.

[0036]FIG. 15. Shows alignment of the E_(p) sequence with Man-1-PGuanylyltransferases.

[0037]FIG. 16. Shows alignment of the E_(p) sequence with NAcGlc-1-PUridylyltransferases.

[0038]FIG. 17. Shows alignment of the E_(p) sequence with Glc-1-PCytidylyltransferases.

[0039]FIG. 18. Shows general Nucleotidylyltransferase Alignment.

[0040]FIG. 19. FIG. 19 is a BLASR analysis for E_(p) seqences, showingsequences producing high-scoring segment pairs.

[0041]FIG. 20. FIGS. 20(a) and 20(b) depict NDP-sugar nucleotides thatmay be prepared using nucleotydylyl-transferases as enzymes inaccordance with the present invention.

[0042]FIG. 21. Interaction between Ep and the glucose moiety in thesugar binding pocket.

[0043]FIG. 22. Summary of sugar phosphate accepted by Ep and mutants

[0044]FIG. 23. One dimensional representation of FIG. 21 illustratingsome of the important contacts and potential sites for engineeringpromiscuity of nucleotidyly-transferases.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present inventors discovered that the Salmonella enterica LT2rmlA-encoded α-D-glucopyranosyl phosphate thymidylyltransferase (E_(p)),(also referred to as dTDP-glucose synthase, dTDP-glucosepyrophosphorylase, thymidine diphosphoglucose pyrophosphorylase andthymidine diphosphate glucose pyrophosphorylase), which catalyzes theconversion of α-D-glucopyranosyl phosphate (Glc-1-P) and dTTP todTDP-α-D-glucose (TDP-Glc) and pyrophosphate (PP_(i)), displaysunexpected promiscuity toward both its nucleotide triphosphate (NTP) andits sugar phosphate substrates. Through a substrate specificityreevaluation of Salmonella enterica LT2 α-D-glucopyranosyl phosphatethymidylyltransferase (E_(p)), the present inventors made the surprisingdiscovery that this enzyme can convert a wide variety of phosphates,including for example, α-D-hexopyranosyl phosphates, including, but notlimited to, deoxy-α-D-glucopyranosyl, aminodeoxy-α-D-hexopyranosyl andacetamidodeoxy-α-D-hexopyranosyl phosphates to their corresponding dTDP-and UDP-nucleotide sugars.

[0046] This discovery led to the invention by the present inventors ofgeneral chemo-enzymatic methods of rapidly generating nucleotidediphosphosugar reagents. These methods allow for the provision of asubstrate set for developing in vitro glycosylation systems, which areuseful for, inter alia, in vitro production of known bioactivemetabolites and of new bioactive metabolites.

[0047] α-D-Hexopyranosyl Phosphates and Methods of Making the Same

[0048] An embodiment of the invention includes α-D-hexopyranosylphosphates, methods including combining these phosphates with NTP in thepresence of nucleotidylyl-transferase, which may be wild type ormutated, and nucleotide sugars produced by converting such hexopyranosylphosphates using nucleotidylyl-transferases, such as E_(p).

[0049] E_(p) is encoded by rmlA, which was previously known as rfbA(Reeves et al. Trends Microbiol. 1996, 4, 495-502). The rmlA-encodedE_(p) was overexpressed in E. coli to provide the desired E_(p) as >5%of the total soluble protein. The corresponding E_(p) was purified tonear homogeniety with a specific activity of 110 U mg⁻¹, a 2-foldimprovement over the previously reported values. (See Lindquist, L.;Kaiser, R.; Reeves, P. R.; Lindberg, A. A. Eur. J. Biochem., 1993, 211,763-770.) An (NH₄)2SO₄ precipitate of E. coli-prfbA-C crude extracts wasdialyzed against buffer B (20 mM Tris.HCl, 1 mM EDTA, pH 7.5) Thedialysate was resolved by anion exchange (DE52, 3×15 cm, 50 mL buffer Bwash followed by a linear gradient of 0-500 mM NaCl, 1.0 mL min⁻¹) andthe E_(p) fractions combined, concentrated and further resolved by FPLCgel filtration (S-200, 2×70 cm, 50 mM Tris.HCl, 200 mM NaCl, pH 7.5).The purified E_(p) was stored in aliquots (−80° C.) until used.

[0050] Although α-D-glucopyranosyl phosphate (2) (FIG. 2),α-D-mannopyranosyl phosphate (compound 56) (FIG. 2) andα-D-galactopyranosyl phosphate (57) (FIG. 2) were commercially availablefor examination as potential substrates for E_(p), most of theα-D-hexopyranosyl phosphates examined were synthesized from free sugars.

[0051] For synthetically derived α-D-hexopyranosyl phosphates,particularly glycosyl phosphates, a general phosphorylation strategyfrom the appropriately protected precursor relied upon

[0052] i) anomeric activation via the ethy 1-thio-β-D-pyranoside [toform e.g., Ethyl 2,3,4-tri-O-benzoyl-6-deoxy-1-thio-β-D-glucopyranoside(9), Ethyl 2,3,6-tri-O-benzoyl-4-deoxy-1-thio-β-D-glucopyranoside (17),Ethyl 2,4,6-tri-O-benzoyl-3-deoxy-1-thio-β-D-glucopyranoside (25), Ethyl2,3,4,6-tetra-O-benzoyl-1-thio-β-D-gulopyranoside (30), Ethyl2,3,4,6-tetra-O-benzoyl-1-thio-β-D-allopyranoside (35) and Ethyl3,4,6-tri-O-benzoyl-2-deoxy-1-thio-β-D-glucopyranoside (40) (The α/β-40mixture (1:1.5) was chromatographically resolved.) (FIG. 1(b))],

[0053] ii) deprotection/reprotection [to form e.g., Ethyl2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (10), Ethyl2,3,6-tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside (18), Ethyl2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (26), Ethyl2,3,4,6-tetra-O-benzyl-1-thio-β-D-gulopyranoside (31), and Ethyl2,3,4,6-tetra-O-benzyl-1-thio-β-D-allopyranoside (36) (FIG. 1(b))],

[0054] iii) phosphorylation [to form e.g.,Dibenzyl-(2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl) phosphate (11),Dibenzyl-(2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl) phosphate (19),Dibenzyl-(2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl) phosphate (27),Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-gulopyranosyl) phosphate (32),Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-allopyranosyl) phosphate (37), andDibenzyl-(3,4,6-tri-O-benzoyl-2-deoxy-α-D-glucopyranosyl) phosphate (41)(FIG. 1(b))], and

[0055] iv) complete deprotection [to form e.g., Disodium6-deoxy-α-D-glucopyranosyl phosphate (12), Disodium4-deoxy-α-D-glucopyranosyl phosphate (20), Disodium3-deoxy-α-D-glucopyranosyl phosphate (28), Disodium α-D-gulopyranosylphosphate (33), Disodium α-D-allopyranosyl phosphate (38) and Disodium2-deoxy-α-D-glucopyranosyl phosphate (43) (FIG. 1(b))].

[0056] In FIG. 1(b): (a) Ph₃P, CCl₄; (b) Ac₂O, pyr; (c) (i) LiAlH₄, (ii)AcOH/HCl, (iii) BzCl, pyr; (d) BzCl, pyr; (e) pFPTC-Cl, DMAP; (f)(n-Bu)3SnH; (g)(i) NaH, imidazole; (ii) CS₂; (iii) CH₃I; (h) AIBN,(n-Bu)₃SnH; (i) (i) CF₃CO₂H, (ii) BzCl, pyr; (j) EtS-TMS, ZnI₂; (k) (i)NaOMe; (ii) NaH; (iii) BnBr; (l) (i) (BnO)2P(O)OH, NIS; (m) H₂, Pd/C;(n) (i) HBr; (ii) (BnO)2P(O)OH, silver triflate, 2,4,6-collidine; (O)NaOH; (p) AcOH/HCl. In each case, cation exchange provided the Na+ salt.

[0057] The overall yield of this four-step phosphorylation strategyranged from 19%-28% including the final ion exchange. FIG. 1(b) showsthese glycosyl phosphates and methods for synthesizing them. Theseglycosyl phosphates, and methods for making them, are provided by thepresent invention.

[0058] The present method includes anomerically activating an ethyl1-thio-β-D-pyranoside to form a compound having the formula

[0059] wherein R² is OCH₃, OBz, or OH,

[0060] R³ is OH, OAc, or OBz,

[0061] R⁴ is H, OH, or a halogen atom, and

[0062] R⁵, R⁶, R⁷, R⁸, and R⁹ are each OBz,

[0063] and three or more of R³, R⁵, R⁶, R⁷, R⁸, and R⁹ are OBzsubstituents; deprotecting the OBz substituents to convert at least onesuch substituent to a OBn substituent; phosphorylating to form acompound of the formula

[0064] wherein R¹ is OCH₃, OBz, OAc or OH,

[0065] R² is OCH₃, OBz, OAc or OH,

[0066] R³ is OH, OAc, or OBz,

[0067] R⁴ is H, OH, OBz, OAc or a halogen atom,

[0068] R⁵, R⁶, R⁷, R⁸, and R⁹ are each OBz or OAc, and R¹⁰ is OH, orOBn,

[0069] wherein at least four of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ areindependently OBn or OBz substituents; and

[0070] deprotecting to convert any OBn substituents to OH substituents.

[0071] Preferably, the α-D-hexopyranosyl phosphate is a glycosylphosphate. Also included are α-D-hexopyranosyl phosphates, preferablyglycosyl phosphates synthesized by these methods. Preferably theseα-D-hexopyranosyl phosphates are selected from the group consisting ofdeoxy-α-D-glucopyranosyl, aminodeoxy-α-D-hexopyranosyl andacetamidodeoxy-α-D-hexopyranosyl phosphates.

[0072] The present invention also includes a method that includesproviding isolated E_(p) having the formula

[0073] wherein R¹ is OCH₃, OBz, OAc or OH,

[0074] R² is OCH₃, OBz, OAc or OH,

[0075] R³ is OH, OAc, or OBz,

[0076] R⁴ is H, OH, OBz, OAc or a halogen atom,

[0077] R⁵, R, R⁷, R⁸, and R⁹ are each OBz or OAc, and

[0078] R¹⁰ is OH, or OBn,

[0079] wherein at least four of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ areindependently OH or OBz substituents.

[0080] Alternatively, phosphorylation ofDibenzyl-(2,3,4,6-tetra-O-benzoyl-α-D-altropyranosyl) phosphate (45),Dibenzyl-(2,3,4,6-tetra-O-benzoyl-α-D-idopyranosyl) phosphate (49) andDibenzyl-(2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl) phosphate (53) (FIG.1(b)) via the glycosyl halide followed by complete deprotection gave theglycosyl phosphates Disodium α-D-altropyranosyl phosphate (47), Disodiumα-D-idopyranosyl phosphate (51) and Disodium α-D-talopyranosyl phosphate(55) as depicted in FIG. 1(b) in an overall yield ranging from 37%-47%.The 6-deoxy precursor1,2,3,4-tetra-O-benzoyl-6-deoxy-α,β-D-glucopyranose (8) may besynthesized by LiAlH₄ reduction and subsequent benzoylation of thehalide Methyl 2,3,4-tri-O-acetyl-6-chloro-6-deoxy-α-D-glucopyranoside(7). (See Anisuzzaman, A. K. M.; Whistler, R. L. Carbohydr. Res. 1978,61, 511-518.). For the 4-deoxy progenitor, deoxygenation at C-4 may beaccomplished by selective benzoylation of methyl β-D-galactopyranosideMethyl β-D-galactopyranoside (13) (as depicted in FIG. 1(b)) to providethe desired tribenzolated Methyl2,3,6-tri-O-benzoyl-β-D-galactopyranoside (14) (54%) as well as thetetrabenzolated derivative (19%). Subsequent C-4 activation Methyl2,3,6-tri-O-benzoyl-4-O-pentafluorophenoxythiocarbonyl-β-D-galactopyranoside(15) and (n-Bu)₃SnH reductive 4-deoxygenation were accomplished asdescribed in Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O.Carbohydr. Res. 1993, 243, 139-164 to give the desired 4-deoxy precursorMethyl 2,3,6-tri-O-benzoyl-4-deoxy-β-D-galactopyranoside (16). The3-deoxy predecessor 1,2,4,6-tetra-O-benzoyl-3-deoxy-α-D-glucofuranose(24) (FIG. 1(b)) was synthesized from1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (21) by reduction of thepreviously reported furanose1,2:5,6-Di-O-isopropylidene-3-O-(methylthio)thiocarbonyl-α-D-glucofuranose(22) (See Zhiyuan, Z.; Magnusson, G. Carbohydr. Res. 1994, 262, 79-101),while the 2-deoxy precursor (39) derived from a commercial source.

[0081] Thus, another embodiment of the present invention includesmethods of making α-D-hexopyranosyl phosphates, which include, but arenot limited to, phosphorylating a phosphate selected from the groupconsisting of Dibenzyl-(2,3,4,6-tetra-O-benzyol-α-D-altropyranosyl)phosphate, Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-idopyranoxyl)phosphate,and Dibenzyl-(2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl) phosphate via aglycosyl halide; and deprotecting to form a glycosyl phosphate selectedfrom the group conssiting of Disodium α-D-altropyranosyl phosphate,Disodium α-Didopyranosyl phosphate and Disodium α-D-talopyranosylphosphate. The present invention also includes α-D-hexopyranosylphosphates prepared according to this method.

[0082] Nucleotide Sugars and Methods of Synthesizing the Same

[0083] The present invention includes methods of making nucleotidesugars, which include combining α-D-hexopyranosyl phosphate and NTP inthe presence of at least one mutated nucleotidylyltransferase. Othermethods according to the present invention include combiningα-D-hexopyranosyl phosphate and NTP other than TTP in the presence of atleast one nucleotidylyltransferase, and combining NTP andα-D-hexopyranosyl phosphate other than Glc1P in the presence of at leastone nucleotidylyltransferase.

[0084] The present invention includes a method of synthesizingnucleotide sugars that includes combining a nucleotidylyl-transferase,α-D-glucopyranosyl phosphate, Mg⁺², NTP and inorganic pyrophosphatase,and incubating. Preferably, the incubating is at a temperature of fromabout 30° C. to about 45° C., preferably about 33° C. to about 42° C.,even more preferably about 37° C. for about 20 to about 40 minutes,preferably about 25 to about 35 minutes, and even more preferably about30 minutes. The nucleotydylyltransferase according to these methods mayinclude one or more natural and/or mutated nucleotidylyltransferases,such as natural and/or mutated E_(p).

[0085] The present invention further includes nucleotide sugars made bythe methods described herein.

[0086] Nucleotide sugars of the present invention may be selected fromthe group consisting of TDP-sugar, GDP-sugar, CDP-sugar, UDP-sugar, andADP-sugar and combinations thereof.

[0087] In embodiments utilizing or including mutatednucleotidylyltransferases, a preferred mutated nucleotidylyltransferaseis E_(p) mutated at one or more amino acids selected from the groupconsisting of V173, G147, W224, N112, G175, D111, E162, T201, I200,E199, R195, L89, L89T, L109, Y146 and Y177.

[0088] In embodiments utilizing or including mutated nucleotidylyltransferases, a preferred mutated nucleotidylyl transferases is E_(p)mutated at one or more amino acids in its active site, its divalentcation binding site, and/or its auxiliary site.

[0089] Likewise, other preferred mutated nucleotidylyl transferasesinclude nucleotidylyl transferases mutated at one or more amino acids intheir active sites, divalent cation binding sites, and/or theirauxiliary sites.

[0090] To evaluate the synthetic utility of purifiedthymidylyltransferase, E_(p), α-D-hexopyranosyl phosphate, Mg⁺² and NTPwere incubated at about 37° C. for about 30 min and the extent ofproduct formation determined by HPLC. The results of these assays areillustrated in FIG. 2. Confirmation of product formation was based uponHPLC co-elution with commercially available standards and/or HPLCisolation and high resolution mass spectroscopy of the product. (Forselect compounds, product peaks were lyopholized and submitted directlyfor HRMS (FAB) analysis.) As controls, little or no product formationwas observed in the absence of E_(p), glycosyl phosphate, Mg⁺², or NTP.A reaction containing 5 mM NTP, 10 mM sugar phosphate and 5.5 mM MgCl₂in a total volume of 50 μL 50 mM potassium phosphate buffer, pH 7.5 at37° C. was initiated by the addition of 3.52 U E_(p) (1 U=the amount ofprotein needed to produce 1 μmol TDP-D-glucose min⁻¹). The reaction wasincubated with slow agitation for 30 min at 37° C., quenched with MeOH(50 μL), centrifuged (5 min, 14,000×g) and the supernatant was stored at−20° C. until analysis by HPLC. Samples (20 μL) were resolved on aSphereclone 5u SAX column (250×4.6 mm) fitted with a guard column(30×4.6 mm) using a linear gradient (20-60 mM potassium phosphatebuffer, pH 5.0, 1.5 mL min⁻¹, A₂₇₅ nm).

[0091] The following nucleotide sugars are non-limiting examples ofnucleotide sugars according to the present invention, which maypreferably be produced in accordance with one or more of the methodsdescribed herein, and in particular the reactions of FIG. 2: (58)Thymidine 5′-(α-D-glucopyranosyl diphosphate) (HRMS (FAB) calc forC₁₆H₂₅O₁₆N₂P₂ 563.0705; found m/z 563.0679 (M+H)); (59) Uridine5′-(α-D-glucopyranosyl diphosphate) (HRMS (FAB): calc for C₁₄H₂₃O₁₇N₂P₂565.0507; found m/z 565.0472 (M+H)); (60) Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB) calc forC₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0714 (M+H)); (61) Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcC₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0510 (M+H)); (62) Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB) calc forC₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0720 (M+H)); (63) Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcC₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0485 (M+H)); (64) Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB) calc forC₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0693 (M+H)); (65) Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcC₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0500 (M+H)); (66) Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB) calc forC₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0730 (M+H)); (67) Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcC₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0492 (M+H)); (68) Thymidine5′-(α-D-mannopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.0701 (M+H)); (69) Uridine 5′-(α-D-mannopyranosyl diphosphate) (HRMS(FAB): calc 565.0507; found m/z 565.0503 (M+H)); (70) Thymidine5′-(α-D-galactopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; foundm/z 563.0710 (M+H)); (71) Uridine 5′-(α-D-galactopyranosyl diphosphate)(HRMS (FAB): calc 565.0507; found m/z 565.0508 (M+H)); (72) Thymidine5′-(α-D-allopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.0715 (M+H)); (73) Uridine 5′-(α-D-allopyranosyl diphosphate) (HRMS(FAB): calc 565.0507; found m/z 565.0507 (M+H)); (74) Thymidine5′-(α-D-altropyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.0699 (M+H)); (75) Uridine 5′-(α-D-altropyranosyl diphosphate) (HRMS(FAB): calc 565.0507; found m/z 565.0511 (M+H)); (76) Thymidine5′-(α-D-gulopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.00712 (M+H)); (77) Uridine 5′-(α-D-gulopyranosyl diphosphate) (HRMS(FAB): calc 565.0507; found m/z 565.0512 (M+H)); (78) Thymidine5′-(α-D-idopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.0708 (M+H)); (79) Uridine 5′-(α-D-idopyranosyl diphosphate) (HRMS(FAB): calc 565.0507; found m/z 565.0507 (M+H)); (80) Thymidine5′-(α-D-talopyranosyl diphosphate) (HRMS (FAB) calc 563.0705; found m/z563.0710 (M+H)); and (81) Uridine 5′-(α-D-talopyranosyl diphosphate)(HRMS (FAB): calc 565.0507; found m/z 565.0499 (M+H)); although data isnot depicted for all products.

[0092] Other nucleotide sugars in accordance with the present inventioninclude, but are not limited to, the following: (109) Thymidine5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z 562.0837 (M+H)); (110) Uridine5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z 564.0640 (M+H)); (111) Thymidine5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z 562.0848 (M+H)); (112) Uridine5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z 564.0638 (M+H)); (113) Thymidine5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z 562.0835 (M+H)); (114) Uridine5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z 564.0622 (M+H)); (115) Thymidine5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z 562.0842 (M+H)); (116) Uridine5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB): calcfor C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z 564.0630 (M+H)); (117) Thymidine5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB):calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z 604.0953 (M+H)); (118)Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0732 (M+H)); (119)Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z 604.0940 (M+H)); (120)Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0730 (M+H)); (121)Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z 604.0947 (M+H)); (122)Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0735 (M+H)); (123)Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z 604.0951 (M+H)); (124)Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0738 (M+H)); (125)Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₆H₂₆O₁₄N₃P₂ 546.0889; found m/z 546.0895 (M+H)); and(126) Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate)(HRMS (FAB): calc for C₁₅H₂₄O₁₅N₃P₂ 548.0682; found m/z 548.0673 (M+H)).

[0093] Further nucleotide sugars in accordance with the presentinvention include, but are not limited to, the following:

[0094]FIG. 2 illustrates the utiltity of E_(p) as a catalyst/reagent tosimplify the synthesis of useful nucleotide sugars—of the twelveglycosyl phosphates tested (which include all possible α-D-hexoses andmonodeoxy α-D-glucoses), all produce product with both TTP and UTP underthe conditions described. These yields might be further improved byusing pyrophosphatase to drive the equilibrium of the reaction. Anexamination of accepted α-D-hexopyranosyl phosphates with TTP suggeststhat E_(p) prefers pyranosyl phosphates, which are predicted to existpredominately as ⁴C₁ conformers [e.g., (12), (20), (28), (43),α-D-glucopyranosyl phosphate (2), α-D-mannopyranosyl phosphate (56), andα-D-galactopyranosyl phosphate (57) (FIGS. 1 and 2)], while thosepredicted to not adopt the ⁴C₁ conformation [e.g., Ethyl2,3,4,6-tetra-O-benzyl-1-thio-β-D-gulopyranoside (31), α-D-allopyranosylphosphate (38), α-D-altropyranosyl phosphate (47), α-D-idopyranosylphosphate (51) and α-D-talopyranosyl phosphate (55) (FIGS. 1 and 2)]show less activity.

[0095] Regarding specific interactions required for conversion, analysisof the corresponding deoxy series [(12), (20), (28) and (43) (FIGS. 1and 2)] implicates only a single required hydroxyl (C-2), the removal ofwhich impairs the yield by >70%. A similar trend is observed in the UTPseries with two exceptions, glycosides (28) and α-D-mannopyranosylphosphate (56) (FIGS. 1 and 2). Cumulatively, these results suggestthat, while the C-2 hydroxyl is important for turnover, alterations atC-3 in the context of UTP result in adverse cooperativity.

[0096] Aminodeoxy-α-D-hexapyranosyl phosphates andacetamidodeoxy-α-D-hexapyranosyl phosphates are each examples ofα-D-hexapyranosyl phosphates that may be used in accordance with thepresent invention. A direct comparison of theaminodeoxy-α-D-glucopyranosyl phosphate series to their correspondingacetamidodeoxy analogs provides insight pertaining to the ability of theE_(p) active site to accommodate additional steric bulk.

[0097] Of the aminodeoxy-α-D-glucopyranosyl phosphates examined, onlytwo, 2-amino-2-deoxy-α-D-glucopyranosyl phosphate (107) (FIG. 4) and2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate (108) (FIG. 4), werecommercially available. The syntheses of the remaining analogs divergedfrom the key intermediates Ethyl6-azide-2,3′,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (89),Ethyl 4-azide-2,3,6-tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside (94)and Ethyl 3-azide-2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside(100) (FIG. 3 (b)).

[0098] Thus, the present invention includes a method of makingaminodeoxy-α-D-glucopyranosyl phosphates comprising converting anintermediate selected from the group consisting of ethyl6-azide-2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (89),ethyl 4-azide-2,3,6 tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside(94), and ethyl3-azide-2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (100) to acorresponding amide.

[0099] Ethyl 1-thio-β-D-pyranosides (89) and (100) derived frompreviously reported glycosides (FIG. 3(b)(87)) (see V. Maunier, P.Boullanger, D. Lafont, Y. Chevalier, Carbohydr. Res. 1997, 299, 49-57)and FIG. 3(b)(98) (W. A. Greenberg, E. S. Priestley, P. S. Sears, P. B.Alper, C. Rosenbohm, M. Hendrix, S.-C. Hung, C.-H. Wong, J. Am. Chem.Soc. 1999, 121, 6527-6541), respectively), while (94) was synthesized,from the previously reported compound (93) (FIG. 3(b)) (P. J. Garegg, I.Kvarnstrom, A. Niklasson, G. Niklasson, S. C. T. Svensson, J. Carbohydr.Chem. 1993, 12, 933-953) in a manner similar to that of thedeoxy-α-D-glucopyranosyl phosphate syntheses described herein.Specifically, this strategy invoked a protection scheme to selectivelyexpose the position of substitution followed by activation (via TsCl orTf₂O) and SN² displacement by sodium azide. From the divergent point(FIG. 3 (89), (94) and (100)), an efficient azide selective SnCl₂reduction followed by acetylation gave the desired ethyl1-thio-β-D-pyranoside precursors (90), (95), and (101). Finally, thesubsequent phosphorylation of FIG. 3(b) (89), (90), (94), (95), (100),and (101) was accomplished by reaction with dibenzyl phosphate aspreviously described where the culminating reductive deprotection alsoled to the conversion of the FIG. 3(b) (89), (94), and (100) azides tothe desired amines. As an aminodideoxy sugar representative,4-amino-4,6-dideoxy-α-D-glucopyranosyl phosphate (FIG. 3(b) (102)) wasalso synthesized from peracetylated D-fucose (FIG. 3(b) (103)) asillustrated in FIG. 3 using a similar strategy.

[0100] To evaluate the synthetic utility of thymidylyl-transferase,purified E_(p), α-D-glucopyranosyl phosphate, Mg⁺², NTP and inorganicpyrophosphatase were incubated at 37° C. for 30 min and the extent ofproduct formation determined by HPLC. The inorganic pyrophosphatase wasincluded to drive the reaction forward. A reaction containing 2.5 mMNTP, 5.0 mM sugar phosphate, 5.5 mM MgCl₂ and 10 U inorganicpyrophosphatase in a total volume of 50 μL 50 mM potassium phosphatebuffer, pH 7.5 at 37° C. was initiated by the addition of 3.52 U E_(p)(1 U=the amount of protein needed to produce 1 mol TDP-D-glucose min⁻¹).The reaction was incubated with slow agitation for 30 min at 37° C.,quenched with MeOH (50 μL), centrifuged (5 min, 14,000×g) and thesupernatant was stored at −20° C. until analysis by HPLC. Samples (30μL) were resolved on a Sphereclone 5u SAX column (150×4.6 mm) fittedwith a SecurityGuard ™ cartridge (Phenomenex; Torrance, Calif.) using alinear gradient (50-200 mM potassium phosphate buffer, pH 5.0, 1.5 mLmin⁻¹, A₂₇₅ nm).

[0101] The results of these assays are illustrated in FIG. 4. For eachassay, confirmation of product formation was based upon high resolutionmass spectroscopy of HPLC-isolated products and, also in some cases,HPLC co-elution with commercially available standards. (Allostericactivation is common for the nucleotidylyltransferase family (forexamples see: M. X. Wu, J. Preiss, Arch. Biochem. Biophys. 1998, 358,182-188; and D. A. Bulik, P. van Ophem, J. M. Manning, Z. Shen, D. S.Newburg, E. L. Jarroll, J. Biol. Chem. 2000, 275, 14722-14728) althoughdata is not yet available pertaining to the allosteric effectors ofE_(p).) As controls, no product formation was observed in the absence ofE_(p), glucopyranosyl phosphate, Mg⁺², or NTP.

[0102] The nucleotide sugars (109)-(126) set forth above are examples ofnucleotide sugars of the present invention, which may be produced inaccordance with the methods described herein, and in particular thereactions diagramed in FIG. 4. A comparison of theaminodeoxy-α-D-glucopyranosyl phosphate/dTTP assay results (FIG. 4 (85),(91), (96), and (107)) to the E_(p) native reaction (FIG. 4, (2)/dTTP)reveals that amino substitution has little or no effect on productformation, and, with the exception of compound (85) (FIG. 4), a similarphenomenon is observed in presence of UTP.

[0103] The divergence of compound (85) from this trend is consistentwith UTP-dependent E_(p) “adverse cooperativity” in the presence ofcertain hexopyranosyl phosphates, as described herein. This phenomenonis perhaps attributable to allosteric activation by dTTP. Evaluation ofthe acetamidodeoxy-α-D-glucopyranosyl phosphate/dTTP assays (FIG. 4compounds (86), (92), (97) and (108)), in comparison to theirnon-acetylated counterparts (FIG. 4 (85), (91), (96) and (107)), revealthat a bulky N-acetyl group at C-2 or C-3 (FIG. 4 (97) and (108)) iswell-tolerated while the identical C-4 or C-6 substitution (FIG. 4 (92))and (86)) results in less activity. Given that these effects most likelyderive from unfavored steric interactions, it follows that the E_(p)active site is able to accommodate additional C-2/C-3 bulk while stericslimit the allowed C-4/C-6 substitutions.

[0104] Surprisingly, product formation from FIG. 4 (86)/UTP was markedlyincreased (8-fold) in comparison to (86)/dTTP. This is the first exampleto contradict the typical adverse UTP-dependent effect upon yieldsobserved, as illustrated by FIG. 4 compounds (85) and (97). Finally, acomparison of aminodideoxy-α-D-glucopyranosyl phosphate (FIG. 4(102)(The product of this reaction, thymidine5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate), is an importantcritical intermediate in the formation of the calicheamicinaryltetrasaccharide to that of (FIG. 4 (96)) reveals C-6 deoxygenationdoes not effect dTTP-dependent E_(p) catalysis but greatly diminishesUTP-dependent conversion. However, given independent deoxygenation atC-6 or amino substitution at C-4 (FIG. 4 (91)) each has no effect onproduct yield, data from independent substitutions may not be reliablein predicting the effects of multiple substitutions on product yield.

[0105]FIG. 4 illustrates the utiltity of E_(p) as a catalyst/reagent tosimplify the synthesis of useful nucleotide sugar pools—of the ninesubstrate analogs tested, all provide product with dTTP and with dUTPunder the conditions described. Further, seven with dTTP and four withUTP provide appreciable product (>50% conversion) under the conditionsdescribed.

[0106] Nucleotide sugars produced via the exploitation of thepromiscuity of E_(p) include, but are not limited to, compounds(58)-(81), (109)-(126) and those set forth in FIGS. 20(a) and 20(b).

[0107] Nucleotidylyltransferases

[0108] Structure-Based Engineering of E_(p)

[0109] The present inventors have determined the first three dimensionalstructures of this unique enzyme in complex with the productUDP-α-D-glucose (UDP-Glc) and with the substrate dTTP at 1.9 Å and 2.0 Åresolution, respectively. A three dimensional structure of E_(p) isdepicted in FIG. 6. This discovery has facilitated the elucidation ofthe molecular details of E_(p) substrate recognition. The structuresreveal the catalytic mechanism of thymidylyltransferases, which isfurther supported by new kinetic data. The present inventors have alsoused structure-based engineering or mutations of E_(p) to producemodified enzymes. These inventive enzymes are capable of utilizing“unnatural” sugars previously not accepted by wild-type E_(p).

[0110] Structure Determination

[0111] The E_(p)-UDP-Glc structure was determined usingseleno-methionine-labeled protein crystals and a data-set collected at awavelength corresponding to the selenium absorption peak. Arepresentative portion of the experimental electron density is shown inFIG. 6. The E_(p)-dTTP structure was subsequently determined bymolecular replacement using the E_(p)-UDP-Glc monomer structure as asearch model.

[0112] Overview of the E_(p) structure

[0113] The structure of the biologically active E_(p) tetramer isillustrated in FIG. 7. The present model is refined at 2.0 Å resolutionto an R factor of 18.3% with restrained temperature factors and goodstereochemistry. FIG. 7a shows E_(p) in complex with UDP-Glc and FIG. 7bdisplays the E_(p)-dTTP complex. The two tetrameric structures are verysimilar with r.m.s.d. for equivalent C_(α) positions=1.0 Å. The enzymehas overall dimensions of about 80 Å×80 Å×60 Å and a compact tertiarystructure generated by four monomers packing tightly against each otheralong two two-fold axes of symmetry drawn on the leftmost panel of FIG.7. The overall surface area buried during tetramer formation isapproximately 10,000 Å, equivalent to the surface of one monomer. Themonomer interactions are dominated by helix-helix packing of the fourlarge helices in the center of the E_(p) tetramer and surroundingextensive loop-loop interactions involving multiple van der Waalscontacts, hydrogen bonds, and salt bridges. The active site pockets ofthe monomers are located close to, but not overlapping with, the monomerinterface.

[0114] The E_(p) monomer (FIG. 8) is a two-domain molecule with overallsize of approximately 50 Å×50 Å×50 Å. The domain containing the activesite is dominated by a large seven-stranded mixed central β-sheet, withan unusual left-handed twist, packed against three α-helices on one sideand another three α-helices on the other. Its extensive hydrophobic corecontains no cavities and is dominated by aromatic side chains.

[0115] This domain has overall resemblance, including the location ofthe active site in a large pocket on the top of the β-sheet, to othernucleotide binding proteins (see Vrielink, A., Ruger, W., Dreissen, H.P. C. & Freemont, P. S. Crystal Structure of the DNA Modifying Enzymeβ-Glucosyltransferase in the Presence and Absence of the SubstrateUridine Diphosphoglucose, EMBO J. 13, 3413-3422 (1994); Charnock, S. J.& Davies, G. J. Structure of the Nucleotide-Diphospho-Sugar Transferase,SpsA from Bacillus subtilis, in Native and Nucleotide-Complexed Forms.Biochem. 38, 6380-6385 (1999); Gastinel, L. N. Cambillau, C. & Bourne,Y. Crystal Structures of the Bovine 4Galatosyltransferase CatalyticDomain and Its Complex with Uridine Diphosphogalactose, EMBO J. 18,3546-3557 (1999); Ha, S., Walker, D., Shi, Y. & Walker, S. The 1.9 ÅCrystal Structure of Escherichia coli MurG, a Membrane-AssociatedGlycosyltransferase Involved in Peptidoglycan Biosynthesis. Prot. Sci.9, 1045-1052 (2000); and Brown, K., Pompeo, F., Dixon, S.,Mengin-Lecreulx, D., Cambillau, C. & Bourne, Y. Crystal Structure of theBifunctional N-Acetylglucosamine 1-phosphate uridylyltransferase fromEscherichia coli: A Paradigm for the Related PyrophosphorylaseSuperfamily. EMBO J. 18, 4096-4107 (1999)), containing the α/βopen-sheet Rossmann fold. (Rossmann, M. G., et al., Evolutionary andstructural relationship among dehydrogenases, in The Enzymes, I. P. D.Boyyer, Editor. Academic Press: New York. p. 61-102 (1975); and Branden,C. & Tooze, J. Introduction to Protein Structure. New York: GarlanPublishing, Inc. (1991).) The second E_(p) domain (represented by yellowin (FIG. 8), packing tightly to the side of the active-site domain,contains four α-helices and a two-stranded β-sheet and is involved inthe inter-monomer packing interactions forming the E_(p) tetramer.

[0116] Structural Homology to Glycosyltransferases andUridylyltransferases

[0117] The present inventors' elucidation of the structure of E_(p)represents the first such elucidation of a structure of athymidylyltransferase. Comparison of the structure with the contents ofthe FSSP database, (Holm, L. & Sander, C. Touring Protein Fold Spacewith Dali/FSSP. Nucleic Acids Res., 26, 316-319 (1998)) revealed thatthe overall E_(p) fold is different from other previously determinedstructures. The closest structural homologs of E_(p) are the SpsAglycosyltransferase from Bacillus subtilis and the functionally relatedE. coli enzyme GlmU. GlmU is a bifunctional enzyme containingacetyltransferase and uridylyltransferase domains, respectively. FIG. 8illustrates these three proteins, highlighting the structurally similarregions. As expected, the structural homology lies within thenucleotide-sugar binding domains. The active sites of the enzymes arelocated in pockets on top of the large β-sheet, although the precisepositioning differs between glycosyltransferases andnucleotidyltransferases and involves secondary structure elements, whichare not structurally equivalent. The three-dimensional structures of twoother sugar-phosphate transferring enzymes, α-D-galactopyranosylphosphate (Gal-1-P) uridylyltransferase and kanamycinnucleotidyltransferase are known, but do not activate sugars and bothdiffer structurally and functionally from E_(p).

[0118] Active Site Interactions: Substrate and Product Binding

[0119]FIG. 8 shows two 90° views of the E_(p) active site pocket. Inboth of the E_(p)-dTTP and E_(p)-UDP-Glc structures, the experimentalelectron density for the dTTP and UDP-Glc is excellent. E_(p) utilizesboth dTTP and UTP, but not CTP, and FIG. 9a illustrates the structuralbasis for this substrate specificity. Specifically, the exocyclic N3 andO4 ring atoms of both dT and U are hydrogen bonded to Gln83. Inaddition, O4 hydrogen bonds to the main chain N of Gly88 while O2 isbound to the main chain N of Gly11. Finally, the 3′-hydroxyl group ofthe pentose forms a hydrogen bond with Gln27. The substrate dTTP alsomakes extensive van der Waals contacts with Leu9, Leu89 and Leu109,which form a hydrophobic bed for the nucleoside, and position 5 of thepyrimidine base is far enough from any protein atom to allow an easy fitfor the methyl group of dT in the pocket. The phosphate groups of dTTPlie in an extended position firmly held in place by multipleinteractions with the main chain nitrogen atoms of Ser13, Gly14, andThr15, and with the catalytically important Mg⁺² (see below). Theγ-phosphate also makes a hydrogen bond with Thr15 and both the α- andγ-phosphates bind Arg16. The nearby Arg145, Lys163, and Arg195 create afavorable electrostatic environment, but do not interact directly withdTTP.

[0120] The E_(p) product, UDP-Glc, is bound along the diameter of thesurface pocket. The nucleoside sits in the active site in virtually thesame conformation as the substrate dTTP, with the addition of a hydrogenbond between the 2′-hydroxyl of the ribose and the main chain O ofGly11. In the glucose-binding pocket, as illustrated on (NAT) FIG. 5b,the hydroxyl groups O2, O3 and O4 of the glucose moiety are directlyhydrogen-bonded to protein residues, while O6 is bound to E_(p) via awater molecule. Gln162 binds both O2 and O3, the main chain N of Gly147binds both O3 and O4, and the main chain O of Val173 binds O4. The sidechain of Thr201 is also close to both O2 and O3. In addition, fourwell-ordered water molecules, shown on FIG. 5b, bridge E_(p) and theglucose moiety. Leu109, Leu89, and Ile200 make van der Waals contactswith the underside of the hexose ring and Trp224 and Tyr146 close theglucose binding pocket which would prevent bulkier sugars, for exampledisaccharides, from binding. In the E_(p)-UDP-Glc structure, thephosphate groups are now twisted away from their straight conformationin dTTP so that they can connect the nucleoside with the hexose—see alsoFIG. 10. The phosphates are also much more solvent exposed and do notinteract with any main chain atoms, but instead, with the side chains ofthe positively charged Arg16, Lys163, and Arg195, as well as with watermolecules.

[0121] Divalent Cation Binding Site

[0122] The activity of nucleotidyltransferases is strictly dependent ona divalent cation involved in catalysis via stabilizing the leavingPP_(i) (See Kornfeld, S. & Glaser, L. J. Biol. Chem. 236, 1791-1794(1961)). Crystallographic data generated by the present inventors allowfor the identification of the location of this cofactor and, in thisregion, a Mg⁺² electron density feature, larger than a water moleculeand chemically ideal for a metal location, was modeled. Indeed the Mg⁺²is 2.6 Å away from the β-phosphate oxygen and is also coordinated by theside chain of Gln26, main chain O of Gly11, main chain nitrogens ofSer13 and Gly14, and a water molecule. This region (particularly Gly10to Gly15) is mostly disordered in the E_(p)-UDP-Glc structure,indicating that the Mg⁺², in addition to electrostatically stabilizingthe leaving group, also plays a structural role in folding thesubstrate-binding region of E_(p) around itself to fix the NTP at anoptimal position for the catalytic event.

[0123] A Secondary dTTP-Binding Site and Possible Allosteric Control

[0124] The structure of E_(p)-dTTP, disclosed herein (FIG. 7), indicatesthat the E_(p) tetramer binds eight molecules of dTTP—four in the activesite pockets on top of the β-sheet, and four in an auxiliary sites atthe interface between the subunits. FIG. 9c shows a close-up of a dTTPmolecule in the auxiliary site. There are fewer contacts between E_(p)and dTTP here than in the active site. As a result, CTP, which is notaccepted by E_(p), could easily fit in the auxiliary site. The dTTP baseand the ribose in the secondary site interact with one E_(p) monomer,including hydrogen bonds to the main chain N of Gly116 and Ser152, andvan der Waals contacts with Leu46, Tyr115 and Ile249. The dTTPphosphates, on the other hand, interact primarily with residues of anadjacent E_(p) monomer, including Arg220 and Gly221.

[0125] Several other nucleotidylyltransferases are under allostericcontrol by metabolites distinct from their products or substrates. Thepresence of an auxilary site strongly suggests that E_(p) is also underallosteric control. Indeed, binding of an effector in this hydrophobicpocket at the monomer interface could alter the relative orientation ofthe E_(p) monomers, thus altering the conformation or the access to theactive site. Given the non-specific nature of the observed interactions,and the fact that nucleotidylyl-transferase effectors are generally notsubstrates, the putative E_(p) allosteric effector is most likely notdTTP.

[0126] The E_(p) Catalytic Mechanism

[0127] Before the present experiments, two conflicting hypotheses fornucleotidylyltransferase catalysis were suggested. Lindquist andco-workers proposed a ping-pong bi-bi mechanism for E_(p), the necessaryprerequisite for which is the formation of an enzyme-substrate covalentintermediate. (See Lindquist, L., Kaiser, R., Reeves, P. R. andLindberg, A. A., Purification, Characterization and HPLC Assay ofSalmonella Glucose-1-Phosphate Thymidylylphospherase from the clonedrfbA Gene, Eur. J. Biochem, 211, 763-770 (1993). Alternatively, in arelated enzyme, Frey and co-workers had previously presented evidencefor inverted geometry about the α-phosphate upon attack by Glc-1-P whichled the authors to propose a single displacement mechanism for theentire nucleotidylytransferase family. (Sheu, K.-F. R., Richard, J. P. &Frey, P. A. Stereochemical Courses of Nucleotidyl-transferase andPhosphotransferase Action. Uridine Diphosphate GlucosePyrophosphorylase, Galactose-1-Phosphate Uridylyltransferase, AdenylateKinase, and Nucleoside Diphosphate Kinase Biochem. 18, 5548-5556(1979)).

[0128] In the present invention, a comparison of the topology of theE_(p)-bound substrate (dTTP) to the E_(p)-bound product (UDP-Glc) (FIG.10a) suggests that the Glc-1-P oxygen nucleophile must directly attackthe α-phosphate of dTTP. In this reaction, the formation of aphosphodiester bond on one side of the α-phosphate atom is simultaneouswith the breaking of a phosphodiester bond on the opposing face (to givePP_(i) as the leaving group). Consistent with an S_(N)2 type mechanism,the bond undergoing formation in the structure disclosed herein is“in-line” or 180 degrees away from the leaving group and thus, the twooxygen atoms bonded to the phosphate invert their geometry upon bondformation. Although the α-phosphate here is not chiral, both thereactant (substrate) and product topologies, as well as the architectureof the active site, clearly suggest that an inversion has occurred.

[0129] The present inventors evaluated the E_(p) steady state kineticsin order to further probe the enzymatic mechanism. The intersectingpatterns observed in FIG. 10b and FIG. 10d are consistent with thestructural data in support of a single displacement mechanism ratherthan the previously postulated ping-pong bi bi (double displacement)mechanism. Finally, the E_(p)-dTTP crystals were soaked in a solutioncontaining 2 mM of either Glc-1-P or D-Glc, in addition to the 2 mM dTTPand Mg⁺² already present. The glucose soaks did not significantly alterthe electron density in the active site. On the other hand, Glc-1-Psoaks quickly caused deterioration of the crystal diffraction quality.Data collected with crystals soaked for 30 min revealed electron densityin the active site that was an average of the density in our E_(p)-dTTPand EP-UDP-Glc crystals. Therefore, the phosphate of Glc-1-P isnecessary for binding by E_(p), and the lack of any observable E_(p)-UMPcovalent intermediate in these experiments further supports the singledisplacement mechanism.

[0130] Active-Site Engineering

[0131] Two sugar phosphates not utilized by wild-type E_(p) and twoadditional sugar phosphates poorly utilized by the enzyme were selectedto test rational engineering of E_(p) substrate promiscuity.Specifically, 6-acetamido-6-deoxy-α-D-glucopyranosyl phosphate (FIG. 11(86)) is not well-accepted and α-D-glucopyranuronic acid 1-(dihydrogenphosphate) (FIG. 11 (127)) is not accepted by E_(p), and2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate (FIG. 11 (108)) andα-D-allopyranosyl phosphate (FIG. 11 (38)) lead to poor conversion.Because a representative “unnatural” sugar phosphate was believed to beefficiently converted only by wild-type E_(p),4-amino-4,6-dideoxy-α-D-glucopyranosyl phosphate (FIG. 11 (102)) wasalso tested with all mutants. Sugar phosphates not utilized by awild-type enzyme, e.g., E_(p), and sugar phosphates poorly utilized bythe enzyme may be referred to herein as “unnatural” with respect to thatenzyme.

[0132] Structure-based modeling reveals steric and/or electrostaticinfringements may be the limiting factor in the conversion of“unnatural” sugar phosphates. In an attempt to relieve theseconstraints, three mutants were constructed. In particular, a Thr201Alamutant and Glu162Asp were believed to decrease the steric interferenceat the sugar positions C-2 and/or C-3 for compounds (108) and (38),while a Trp224His substitution was designed to decrease stericconstraints at C-6 of the substrate (e.g. compound (86)). Furthermore,the glucuronic acid derivative (127) offers the unique challenge ofengineering electrostatic balance and the Trp224His variant waspredicted to provide a partial positive charge to assist in(127)-binding in addition to steric relief. Alternatively, Asp-111 (6 Åfrom the substrate C-6-OH) was predicted to result in the electrostaticrepulsion of substrates containing a negative charge at the C-6 of thesugar phosphate. Thus, an additional mutant (Asp111Asn) was constructedto eliminate this effect.

[0133] As a rapid means to assay the entire pool of the four newlyconstructed mutants, the mutants were combined and the mixture directlytested for ability to convert compounds (2), (108), (89), (102), (38),and (127). FIG. 11 shows that the mutant pool was able to turn over allbut one (5) of the sugar phosphates tested. Those substrates turned overinclude (86) and (127), the two sugar phosphates not accepted or poorlyaccepted by wild-type E_(p).

[0134] The following nucleotide sugars were produced by the reactions ofFIG. 11: (117) Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (HRMS (FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z604.0953 (M+H)); (118) Uridine5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB):calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0732 (M+H)); (130)Thymidine 5′-(α-D-glucopyran-6-uronic acid diphosphate) (HRMS (FAB):calc for C₁₆H₂₃O₁₇N₂P₂ 577.0472; found m/z 577.0465 (M+H)); (131)Uridine 5′-(α-D-glucopyran-6-uronic acid diphosphate) (HRMS (FAB): calcfor C₁₅H₂₁O₁₈N₂P₂ 579.2774; found m/z 579.2775 (M+H)); (123) Thymidine5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS (FAB):calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z 604.0951 (M+H)), (124)Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z 606.0738 (M+H)); (125)Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (HRMS(FAB): calc for C₁₆H₂₆O₁₄N₃P₂ 546.0889; found m/z 546.0895 (M+H)); (126)Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc for C₁₅H₂₄O₁₅N₃P₂ 548.0682; found m/z 548.0673 (M+H))although data is not depicted for all products.

[0135] A deconvolution of the mutant pool, by individual mutantanalysis, revealed the Trp224His mutation as responsible for convertingboth (86) and (127). Thr102Ala, on the other hand, was responsible forthe 2-fold increase in the conversion of (108). The remaining twomutants (Asp41Asn and Glu162Asp, not shown in FIG. 11) failed to enhanceconversion, over wild-type E_(p), of any of the tested putativesubstrates. Yet, cumulatively, this small set of directed mutants wasable to successfully turn over three of four targeted “unnatural”substrates. Of particular interest is the Trp224His mutant, whichdisplays enhanced promiscuity without affecting wild-type traits. ThisE_(p) variant will serve as an excellent foundation for secondgeneration double mutants. Finally, the demonstrated ability to testmutant sets via pooling will rapidly expedite the development of thismethodology.

[0136] In E_(p), amino acids that make contacts or near contacts to thesugar in the active site include V173, G147, W224, N112, G175, D111,E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and Y177. Theseamino acids may be mutated in order to alter the specificity of E_(p),as demonstrated herein. Any mutation that alters the specificity may bemade and tested, as taught herein, to determine its effect on thespecificity of E_(p) for its substrate and the efficiency of conversionof substrate to product.

[0137] Thus, the present invention includes a nucleotidylyl-transferasemutated at one or more amino acids selected from the group consisting ofV173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195, L89,L89T, L109, Y146 and Y177. Preferably the nucleotidylyltransferase isE_(p).

[0138] An embodiment of the present invention is directed to anucleotidylyltransferase mutated such that it is capable of having adifferent substrate specificity than a non-mutatednucleotidylyltransferase. Examples include nucleotidylyl-transferaseshaving a substrate specificity for GTP, ATP, CTP, TTP or UTP. Furtherprovided are methods of altering nucleotidylyltransferase substratespecificity comprising mutating the nucleotidylyltransferase at one ormore amino acids selected from the group consisting of V173, G147, W224,N112, G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146and Y177. Preferably the nucleotidylyl-transferase is E_(p). Alsoprovided are nucleotidylyl-transferases, so modified.

[0139] The present invention also includes purine or pyrimidinetriphosphate type nucleotidylyltransferases set forth in FIG. 19, andpurine or pyrimidine triphosphate type nucleotidylyl-transferasesmutated at one or more amino acids selected from the group consisting ofV173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195, L89,L89T, L109, Y146 and Y177.

[0140] Further, sequence comparison reveals that manynucleotidyltransferases bear high degrees of sequence identity to E_(p).The substrate specificity of such enzymes may be altered, using methodsdescribed herein for E_(p), at amino acids that make contacts or nearcontacts to the sugar in the active site. These amino acids may belocated via sequence comparison with E_(p)—the contact sites will oftenbe those at the same relative position as V173, G147, W224, N112, G175,D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 and Y177 inE_(p). FIG. 19 provides a list of nucleotidyltransferases that bear highdegrees of sequence identity to E_(p). FIGS. 12 to 18 show the alignmentof the E_(p) sequence and those of other representativenucleotidyltransferases. Other nucleotidylyltransferases may also bemutated at one or more amino acids in their active sites, divalentcation binding sites and/or auxiliary sites.

[0141] Methods for mutating proteins are well-known in the art. For thepresent invention, it is preferable to perform site-directed mutagenesison the nucleotide encoding the enzyme of interest. In this manner, andusing the guidance provided herein, one of skill in the art can makemutations to the codons encoding the amino acids at the sites of theenzyme desired to be changed. Likewise, the use of site directedmutagenesis allows the worker to ensure that each codon desired to bechanged is changed to encode a different amino acid from the wild-typemolecule. In contrast, the use of random mutagenesis might result inmutated codons encoding the same amino acids as the wild-type codons,due to the degeneracy of the genetic code. Methods for manipulation andmutation of nucleotides, as well as for the expression of recombinantpeptides are well known in the art, as exemplified by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, 1989

[0142] References for nucleotidyltransferases in these Figures, include:AAB31755-Glc-1-P Cytitdylyltransferase from Yersinia pseudotuberculosis.See Thorson J S, Lo S F, Ploux O, He X & Liu H W J. Bacteriol. _(—)176:_(—)5483-5493 (1994) [94350832]; AAC39498-Man-1-P Guanylyltransferasefrom Hypocrea jecorina. Kruszewska, J. S., Saloheimo, M., Penttila, M. &Palamarczyk, G. Direct Submission; B72403- Glc-1-P adenylyltransferasefrom Thermotoga maritima (strain MSB8). Nelson K E, Clayton R A, Gill SR, Gwinn M L, Dodson R J, Haft D H, Hickey E K, Peterson J D, Nelson WC, Ketchum K A, McDonald L, Utterback T R, Malek J A, Linher K D,Garrett M M, Stewart A M, Cotton M D, Pratt M S, Phillips C A,Richardson D, Heidelberg J, Sutton G G, Fleischmann R D, Eisen J A,Fraser C M & et al Nature _(—)399:_(—)323-329 (1999) [99287316];BAA34807-Man-1-P Guanylyltransferase from Candida albicans. Ohta, A. &Sudoh, M. Direct Submission; CAA06172-Glc-1-P Uridiylyltransferase fromStreptococcus pneumoniae. Mollerach M, Lopez R & Garcia E J. Exp. Med._(—)188: _(—)2047-2056 (1998) [99059828]; D83000-Glc-1-Pthymidylyltransferase from Pseudomonas aeruginosa (strain PAO1).; StoverC K, Pham X Q, Erwin A L, Mizoguchi S D, Warrener P, Hickey M J,Brinkman F S, Hufnagle W O, Kowalik D J, Lagrou M, Garber R L, Goltry L,Tolentino E, Westbrock-Wadman S, Yuan Y, Brody L L, Coulter S N, FolgerK R, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong G K, Wu Z &Paulsen I T Nature _(—)406: _(—)959-964 (19100) [20437337];E72229-N-acetylglucosamine-1-phosphate (NacGlc-1-P) Uridylyl-transferasefrom Thermotoga maritima (strain MSB8). Nelson K E, Clayton R A, Gill SR, Gwinn M L, Dodson R J, Haft D H, Hickey E K, Peterson J D, Nelson WC, Ketchum K A, McDonald L, Utterback T R, Malek J A, Linher K D,Garrett M M, Stewart A M, Cotton M D, Pratt M S, Phillips C A,Richardson D, Heidelberg J, Sutton G G, Fleischmann R D, Eisen J A,Fraser C M & et al Nature _(—)399: _(—)323-329 (1999) [99287316];GalU_MYCGE-Glc-1-P Uridylyltransferase from Mycoplasma genitalium.Fraser C M, Gocayne J D, White O, Adams M D, Clayton R A, Fleischmann RD, Bult C J, Kerlavage A R, Sutton G, Kelley J M & et al Science_(—)270: _(—)397-403 (1995) [96026346]; GCAD_BACSU-NacGlc-1-PUridylyltransferase from Bacillus subtilis. Nilsson D, Hove-Jensen B &Arnvig K Mol. Gen. Genet. _(—)218: _(—)565-571 (1989) [90066361];GLGC_BACSU-Glc-1-P Adenylyltransferase from Bacillus subtilis. Kiel J A,Boels J M, Beldman G & Venema G Mol. Microbiol. _(—)11: _(—)203-218(1994) [94195107]; RFB_SALTY-Glc-1-P Cytidylyltransferase fromSalmonella serovar typhimurium (strain LT2). Jiang X M, Neal B, SantiagoF, Lee S J, Romana L K & Reeves P R Mol. Microbiol. _(—)5: _(—)695-713(1991) [91260454].

[0143] According to one embodiment of the present invention mutations atamino acid L89T were tested. Such mutations increased the yield ofallo-, altro-, talo-, gulo- and ido-derivatives. Wild-type and/or thismutant also led to the production of the new nucleotide sugar compoundsset forth in FIGS. 20(a) and (b). Methods of production of suchcompounds and of the mutant nucleotidylyltransferase are as set forthherein with regard to other compounds and mutantnucleotidylyl-transferase. In particular, the compounds may be producedby synthesizing the corresponding sugar phosphate followed by E_(p)catalyzed conversion of the sugar phosphate to the new products.

[0144] The present invention includes the nucleotide sugars of FIGS.20(a) and 20(b), their corresponding sugar phosphates andnucleotidylyltransferases mutated at L89T, which may convert such sugarphosphates to a nucleotide sugar.

[0145] Glycorandomization of Natural Product-Based Metabolites

[0146] The wild-type glycosyltransferases in secondary metabolism showsignificant flexibility with respect to their NDP-sugar donors. Coupledwith the presented E_(p)-catalyzed production of NDP-sugar donorlibraries and the appropriate aglycon, a diverse library of“glycorandomized” structures based upon a particular natural productscaffold can be rapidly generated.

[0147] Accordingly, the present invention is also directed to nucleotidesugar libraries including two or more of the nucleotide sugars describedherein. More preferably the nucleotide sugars are nucleotide sugars madeby the methods described herein, preferably using a natural or mutatednucleotidylyltransferase as a catalyst. The present invention alsoincludes in vitro glycorandomization using such sugar libraries.

[0148] Exploiting the promiscuity of wild type E_(p) and utilizing theability conferred by the methods of the present invention to rationallyengineer variants able to utilize sugar phosphates not previouslyusable, libraries of previously unavailable nucleotide sugars may begenerated. The ability to generate a set of E_(p) variants provides thesubsequent ability to generate, in a simple one pot reaction, diverselibraries of NDP-sugars. Both sugars that were unknown prior to thepresent invention and those that could not be synthesized in vitro priorto the present invention may be synthesized using the methods of thepresent invention. Such libraries of NDP-sugars, in conjunction withdownstream glycosyltransferases, form the basis for the in vitromanipulation of metabolite sugar ligands in a combinatorial fashion (or“glycorandomization”).

[0149] For example, a diverse library of “glycorandomized” structuresbased upon the known antitumor agent mithramycin (FIG. 5) may beconstructed. Beginning with a small pool of sugar phosphates, e.g., 25different sugar phosphates, the anticipated library size would be theresult of combining 25 different sugars at 5 different positions onmithramycin to give 25⁵, or >9.7 million, distinct mithramycin-basedvariants. Furthermore, as alterations of the carbohydrate ligands ofbiologically active metabolites can lead to drastically differentpharmacological and/or biological properties, this approach hassignificant potential for drug discovery. As an example of alterationsof the carbohydrate ligands of biologically active metabolites producingdramatically different pharmacological properties, the structure of4-epidoxorubicin differs from that of doxorubicin, which is more toxic,only in carbohydrate ligands. As an example of alterations of thecarbohydrate ligands of biologically active metabolites producingdramatically different biological properties, the structure oferythromycin, an antibiotic, differs from that of megalomicin, acompound with antiviral and antiparasitic activity, only in carbohydrateligands.

[0150] An embodiment of the invention includes incubating aglycotransferase with one or more of the sugars of a nucleotide sugarlibrary according to the present invention, and a molecule capable ofbeing glycosylated.

[0151] The present inventors have discovered that E_(p) is pliable interms of its substrate specificity. The present inventors have alsodiscovered the three dimensional structure of E_(p) and the moleculardetails of E_(p) substrate recognition. The present inventors haveinvented methods of engineering or modifying E_(p) to vary itsspecificity in a directed manner, conferring the ability to rationallyengineer variants able to utilize sugar phosphates not previouslyusable. The present inventors have also invented a method for thesynthesis of desired nucleotide sugars using both natural and engineeredE_(p). Thus, the present invention will likely broadly impact efforts tounderstand and exploit the biosynthesis of glycosylated bioactivenatural products, many of which are pharmacologically useful. Theability conferred by the methods of the present invention to alternucleotidylyltransferase specificity by design allows the creation ofpromiscuous in vitro systems, which could provide large and diverselibraries of potentially new bioactive metabolites.

[0152] The present invention will now be illustrated by the followingexamples, which show how certain specific representative embodiments ofthe compounds and methods of the present invention, the compounds,intermediates, process steps, and the like being understood as examplesthat are intended to be illustrative only. In particular, the inventionis not intended to be limited to the conditions, order of the steps andthe like specifically recited herein. Rather, the Examples are intendedto be illustrative only.

EXAMPLES

[0153] General Methods.

[0154] Infrared spectra were recorded on a Perkin Elmer 1600 series FTIRspectrophotometer. ¹H NMR spectra were obtained on a Bruker AMX 400 (400MHz) and are reported in parts per million (δ) relative to eithertetramethylsilane (0.00 ppm) or CDCl₃ (7.25 ppm) for spectra run inCDCl₃ and D₂O (4.82 ppm) or CD₃OD (3.35 ppm) for spectra run in D₂O.Coupling constants (J) are reported in hertz. ¹³C NMR are reported in δrelative to CDCl₃ (77.00 ppm) or CD₃OD (49.05 ppm) as an internalreference and ³¹P NMR spectra are reported in δ relative to H₃PO₄ (0.00ppm in D₂O). Routine mass spectra were recorded on a PE SCIEX API 100LC/MS mass spectrometer and HRMS was accomplished by the University ofCalifornia, Riverside Mass Spectrometry Facility. Optical rotations wererecorded on a Jasco DIP-370 polarimeter using a 1.0 or 0.5 dm cell atthe room temperature (25° C.) and the reported concentrations. Meltingpoints were measured with Electrothermal 1A-9100 digital melting pointinstrument. Chemicals used were reagent grade and used as suppliedexcept where noted. ‘Analytical TLC was performed on either E. Mercksilica gel 60 F₂₅₄ plates (0.25 mm) or Whatman AL Sil G/UV silica gel 60plates. Compounds were visualized by spraying I₂/KI/H₂SO₄ or by dippingthe plates in a cerium sulfate-ammonium molybdate solution followed byheating. Liquid column chromatography was performed using forced flow ofthe indicated solvent on E. Merck silica gel 60 (40-63 μm) and highpressure liquid chromatography was performed on a RAININ Dynamax SD-200controlled with Dynamax HPLC software.

[0155] Although the above methods of IR, NMR, Mass Spec, and HRMS werethose used in the examples of the present invention, it would be knownto those skilled in the art that other instruments, conditions, types oftechniques, and the like may be used to visualize compounds, identifycompounds and determine their concentrations and purity.

[0156] Methyl 2,3,4-tri-O-acetyl-6-chloro-6-deoxy-α-D-glucopyranoside(7).

[0157] Compound 7 was prepared as previously described from methylα-D-glucopyranoside (5), (7.26 g, 27.7 mmol) in 82% yeild (Anisuzzaman,A. K. M.; Whistler, R. L. Carbohydr. Res. 1978, 61, 511-518). R_(f)=0.34(2:1 hexane/EtOAc); [α]_(D)=147° (c=1, CHCl₃); ¹H NMR (CDCl₃) 5.46 (t,1H, J=9.5 Hz), 4.98 (m, 2H), 4.02 (m, 1H), 3.82 (dd, 1H, J=12.0, 2.5Hz), 3.73 (dd, 1H, J=12.0, 6.5 Hz), 3.43 (s, 3H), 2.06 (s, 3H), 2.04 (s,3H), 1.99 (s, 3H); ¹³C NMR (CDCl₃) 170.45, 170.42, 169.96, 96.98, 77.67,71.08, 70.46, 70.32, 69.13, 55.85, 43.81, 21.07, 21.05. MS: calcd forC₁₃H₁₉O₈ClNa 360.9, found m/z 360.9 (M+Na).

[0158] 1,2,3,4-tetra-O-benzoyl-6-deoxy-α,β-D-glucopyranose (8).

[0159] Compound 7 (2.9 g, 8.57 mmol) was dissolved in 100 mL dry THF and1.0 g LiAlH₄ slowly added. The corresponding mixture was refluxed for 10hr under argon and the reaction quenched with 10 mL MeOH andconcentrated. The concentrate was then dissolved in a mixture of 40 mLacetic acid and 10 mL 1N HCl and the reaction stirred at 95° C. for 10hrs. The reaction was neutralized with 1N NaOH and the organicsconcentrated, dried over MgSO₄ and purified by silica gel chromatography(4:1 CHCl₃/MeOH). The resulting product was dissolved in 50 mL drypyridine, 8.0 mL benzoyl chloride (68.9 mmol) was added and the reactionstirred overnight at room temperature. To the reaction mixture was addedto 100 mL saturated NaHCO₃ solution and the mixture extracted with CHCl₃(3×100 mL). The combined organics were washed with H₂O (50 mL), brine(50 mL), dried over Na₂SO₄, concentrated and purified by silica gelchromatography (2:1 hexane/EtOAc) to give 2.8 g (56.2%) of the desiredproduct 8 (α/β=3:2). This mixture was utilized directly for the nextstep without further resolution. MS: calcd for C₃₄H₂₈O₉Na 630.2, foundm/z 630.2 (M+Na).

[0160] Methyl 2,3,6-tri-O-benzoyl-β-D-galactopyranoside (14).

[0161] Methyl β-D-galactopyranoside (13), 3.7 g, 19 mmol) gave thedesired product 14 (5.2 g, 54%) and 2.3 g (19%) of the correspondingtetra benzoylated derivative as described in Reist, E. J.; Spencer, R.R.; Calkins, D. F.; Baker, B. R.; Goodman, L. J. Org. Chem. 1965,2312-2317. [α]_(D)=7.3° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.18-7.92 (m, 6H),7.52-7.38 (m, 8H), 5.77 (dd, 1H, J=8.0, 10.4 Hz), 5.37 (dd, 1H, J=3.2,10.3 Hz, 1H), 4.72 (dd, 1H, J=6.6, 11.4 Hz), 4.62 (dd, 1H, J=6.4, 11.4Hz), 4.66 (d, 1H, J=7.9 Hz), 4.36 (m, 1H), 4.08 (t, 1H, J=6.5 Hz), 3.55(s, 3H), 2.50 (br, 1H, ˜OH); ¹³C NMR (CDCl₃): 166.9, 166.3, 165.9,133.9, 133.7, 133.6, 130.4, 130.3, 130.2, 130.1, 130.0, 129.9, 129.4,129.0, 128.9, 128.8, 128.7, 102.6, 74.6, 72.8, 69.9, 67.7, 63.3, 57.3;MS: calcd for C₂₈H₂₆O₉Na 529.1, found m/z 529.0 (M+Na).(Garegg, P. J.;Oscarson, S. Carbohydr. Res. 1985, 137, 270-275.)

[0162] Methyl2,3,6-tri-O-benzoyl-4-O-pentafluorophenoxythiocarbonyl-β-D-galactopyranoside(15).

[0163] Methyl 2,3,6-tri-O-benzoyl-β-D-galactopyranoside (14), (2.3 g,4.5 mmol) gave 2.88 g (86%) purified product 15 as described in Kanie,O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Carbohydr. Res. 1993,243, 139-164. [α]_(D)=9° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.04 (d, 2H, J=7.7Hz), 7.98 (d, 2H, J=7.6 Hz), 7.93 (d, 2H, J=7.7 Hz), 7.58-7.49 (m, 3H),7.44-7.34 (m, 6H), 6.23 (d, 1H, J=3.2 Hz), 5.78 (dd, 1H, J=7.9 Hz), 5.70(dd, 1H, J=3.3, 10.4 Hz), 4.75-4.71 (m, 2H), 4.44 (dd, 1H, J=7.4, 11.0Hz), 4.37 (t, 1H, J=7.0 Hz), 3.57 (s, 3H); ¹³C NMR (CDCl₃) 192.5, 166.3,166.0, 165.5, 134.1, 133.9, 133.7, 130.3, 130.2, 130.1, 129.6, 129.5,128.9, 128.8, 128.6, 102.7, 79.9, 71.3, 71.1, 69.9, 61.5, 57.6; MS:calcd for C₃₅H₂₅O₉SF₅Na 755.1, found m/z 755.1 (M+Na).

[0164] Methyl 2,3,6-tri-O-benzoyl-4-deoxy-β-D-galactopyranoside (16).

[0165] Methyl2,3,6-tri-O-benzoyl-4-O-pentafluorophenoxythiocarbonyl-β-D-galactopyranoside(15), (2.65 g, 3.62 mmol) gave 1.53 g (86%) of the desired compound 16as described in Kanie, O; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O.Carbohydr. Res. 1993, 243, 139-164. [α]_(D)=57.4° (c=1, CHCl₃); ¹H NMR(CDCl₃) 8.07 (d, 2H, J=7.3 Hz), 8.00 (d, 2H, J=7.4 Hz), 7.95 (d, 2H,J=7.3 Hz), 7.58 (t, 1H, J=7.4 Hz), 7.53-7.40 (m, 4H), 7.39-7.34 (m, 4H),5.41 (m, 2H), 4.60 (d, 1H, J=7.5 Hz), 4.51 (dd, 1H, J=5.8, 11.6 Hz),4.46 (dd, 1H, J=4.4, 11.6 Hz), 4.06 (m, 1H), 2.47 (m, 1H), 1.88 (m, 1H);¹³C NMR (CDCl₃) 166.7, 166.3, 165.9, 133.7, 133.6, 133.5, 130.2, 130.1,130.0, 129.7, 128.9, 128.8, 128.7, 102.6, 72.9, 71.9, 70.0, 66.2, 57.4,33.4; MS: calcd for C₂₆H₂₆O₈Na 513.1, found m/z 513.0 (M+Na). (Lin,T.-H.; Kovac, P.; Glaudemans, C. P. J. Carbohydr. Res. 1989, 141,228-238.)

[0166]1,2:5,6-Di-O-isopropylidene-3-O-(methylthio)thiocarbonyl-α-D-glucofuranose(22).

[0167] Compound 22 was prepared as previously described in 93% yield(see Zhiyuan, Z.; Magnusson, G. Carbohydr. Res. 1994, 262, 79-101).[α]_(D)=−34° (c=1, CHCl₃); ¹H NMR (CDCl₃) 5.91 (m, 2H), 4.68 (d, 1H,J=3.77 Hz), 4.31 (m, 1H), 4.10 (dd, 1H, J=5.6, 8.7 Hz), 4.05 (dd, 1H,J=4.6, 8.7 Hz), 2.59 (s, 3H), 1.57 (s, 3H), 1.41 (s, 3H), 1.32 (s, 3H),1.31 (s, 3H); ¹³C NMR (CDCl₃) 112.8, 109.7, 105.4, 84.6, 83.1, 80.1,72.7, 67.3, 27.2, 27.0, 26.6, 25.6, 19.7; MS: calcd for C₁₄H₂₂O₆S₂Na373.0, found m/z 372.8 (M+Na).

[0168] 3-Deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (23).

[0169] To a solution containing 22 (2.6 g, 7.4 mmol) and 120 mg of AIBN(0.73 mmol) in 50 mL dry toluene, 5 mL (n-Bu)₃SnH (18.6 mmol) was addedand the mixture refluxed for 5 hrs under argon. The reaction was thenconcentrated and the residue was applied to a silica gel column(10:1-8:1 hexane/EtOAc) to give 1.58 g substantially pure product 23(87%). [α]_(D)=−9.2° (c=1, CHCl₃); ¹H NMR (CDCl₃) 5.82 (d, 1H, J=3.7Hz), 4.76 (t, 1H, J=4.2 Hz), 4.19-4.07 (m, 3H), 3.84 (m, 1H), 2.18 (dd,1H, J=3.9, 13.2 Hz), 1.77 (m, 1H), 1.51 (s, 3H), 1.42 (s, 3H), 1.35 (s,3H), 1.30 (s, 3H); ¹³C NMR (CDCl₃) 109.6, 107.9, 104.0, 79.4, 77.0,75.7, 65.6, 33.7, 25.7, 24.9, 24.5, 23.6; MS: calcd for C₁₂H₂₀O₅Na267.1, found m/z 266.8 (M+Na). (Barton, D. H. R.; McCombie, S. W. J.Chem. Soc. Perkin Trans. 1 1975, 1574.)²²

[0170]1,2,4,6-tetra-O-benzoyl-3-deoxy-α-D-glucofuranose (24).

[0171] Compound 23 (0.59 g, 2.4 mmol) was treated with a mixture of 9 mLCF₃CO₂H and 1 mL of water for 2 hours at 25° C. The reaction wasconcentrated under reduced pressure, coevaporated with water (2×5 mL)and further dried under vacuum. This material was dissolved in 20 mL ofanhydrous pyridine, to which 2.2 mL (19.3 mmol) of benzoyl chloride wasadded. The mixture was stirred for 10 hr, pyridine removed in vacuo andthe remaining oil diluted with 200 mL EtOAc. The organics washed withsaturated NaHCO₃ (50 mL), water (40 mL), brine (40 mL), dried overNa₂SO₄, and purified with silica gel chromatography (3:1 hexanes/EtOAc)to give 0.89 g product which was used directly without furthercharacterization.

[0172] General Strategy for Formation of Protected Ethyl1-thio-β-D-hexopyranosides.

[0173] Protected ethyl 1-thio-β-D-hexopyranosides may be formed inaccordance with the present invention by the following reaction. Amixture of protected monosaccharide, (ethylthio)-trimethylsilane, andzinc iodide are refluxed for 1½ to 2½ hrs. The reaction is then cooled,diluted, washed, preferably with saturated NaHCO₃ solution, water, andthen brine. The organics are then dried, and preferably concentrated andresolved to give the desired product. Other conditions, reagents, methodsteps, solutions and the like of the present method, may be used inaccordance with the present invention.

[0174] In a typical reaction, a mixture of 3 mmol protectedmonosaccharide, 1.5 mL (ethylthio)trimethylsilane (9.2 mmol) and 1.95 gzinc iodide (6.1 mmol) in 30 mL dry dichloromethane was refluxed for 2hrs under argon atmosphere. The reaction was then cooled and dilutedwith 200 mL CH₂Cl₂, washed successively with saturated NaHCO₃ solution(2×30 mL), water (30 mL) and brine (30 mL). The organics were dried overNa₂SO₄, concentrated and resolved by silica gel chromatography (8:1hexanes/EtOAc) to give the desired product.

[0175] Ethyl 2,3,4-tri-O-benzoyl-6deoxy-1-thio-β-D-glucopyranoside (9).

[0176] Compound 8 (1 g, 1.72 mmol) gave 731 mg (81.5%) of the desiredproduct. R_(f)=0.56 (2:1 hexane/EtOAc); [α]_(D)=7° (c=1.0, CHCl₃); ¹HNMR (CDCl₃) 8.00-7.94 (m, 4H), 7.82 (dd, 1H, J=1.4, 7.1 Hz), 7.52 (m,2H), 7.42-7.37 (m, 5H), 7.23 (m, 2H), 5.85 (t, 1H, J=9.6 Hz), 5.54 (t,1H, J=9.7 Hz), 5.35 (t, 1H, J=9.6 Hz), 4.80 (d, 1H, J=9.9 Hz), 4.92 (m,1H), 2.82 (m, 2H), 1.40 (d, 3H, J=6.2 Hz), 1.26 (t, 3H, J=7.4 Hz); ¹³CNMR (CDCl₃) 164.8, 164.4, 164.2, 132.3, 132.2, 132.1, 128.8, 128.7,128.6, 128.2, 128.0, 127.9, 127.4, 127.3, 127.2, 82.3, 73.9, 73.1, 72.7,69.8, 22.9, 16.8, 13.7; MS: calcd for C₂₉H₂₈O₇SNa 543.1, found m/z 542.9(M+Na).

[0177] Ethyl 2,3,6-tri-O-benzoyl-4-deoxy-1-thio-β-D-glucopyranoside(17).

[0178] Compound 16 (1.5 g, 3.06 mmol) gave 1.24 g desired product(77.8%). [α]_(D)=56.9° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.08 (d, 2H, J=8.0Hz), 8.00 (d, 2H, J=8.2 Hz), 7.96 (d, 2H, J=8.0 Hz), 7.60 (t, 1H, J=6.9Hz), 7.55-7.48 (m, 4H), 7.42-7.36 (m, 4H), 5.50-5.44 (m, 2H), 4.76 (d,1H, J=9.0 Hz), 4.51 (dd, 1H, J=5.7, 1.9 Hz), 4.46 (dd, 1H, J=4.4, 11.9Hz), 4.12 (m, 1H), 2.84-2.69 (m, 2H), 2.53 (m, 1H), 1.91 (m, 1H), 1.27(t, 3H, J=7.6 Hz); ¹³C NMR (CDCl₃) 166.6, 166.2, 165.9, 133.7, 133.6,130.2, 130.1, 129.8, 129.7, 128.8. 184.2, 74.0, 73.0, 71.5, 66.3, 33.6,24.7, 15.4; MS: calcd for C₂₉H₂₈O₇SNa 543.1, found m/z 543.1 (M+Na).

[0179] Ethyl 2,4,6-tri-O-benzoyl-3-deoxy-1-thio-β-D-glucopyranoside(25).

[0180] Compound 24 (0.89 g, 1.5 mmol) gave 0.79 substantially pureproduct (90%). ¹H NMR (CDCl₃) 8.14-7.96 (m, 6H), 7.63-7.40 (m, 9H),5.32-5.21 (m, 2H), 4.79 (d, 1H, J=9.7 Hz), 4.67 (dd, 1H, J=2.9, 12.0Hz), 4.46 (dd, 1H, J=6.0, 12.0 Hz), 4.09 (m, 1H), 2.96 (m, 1H), 2.78 (m,2H), 2.00 (m, 1H), 1.27 (t, 3H, J=7.4 Hz); MS: calcd for C₂₉H₂₈O₇SNa543.1, found m/z 543.1 (M+Na).

[0181] Ethyl 3,4,6-tri-O-benzoyl-2-deoxy-1-thio-β-D-glucopyranoside(40).

[0182] Compound 39 (1.72 g, 2.96 mmol) gave two products, 0.74 g thedesired β isomer (48% yield) and 0.5 g the β isomer (32% yield). βisomer: [α]_(D)=120° (c=1, CHCl₃); IR:_(—)2962, 2871, 1723, 1601, 1450,1314, 1270, 1107, 708, 686 cm⁻¹; ¹H NMR (CDCl₃) 8.06-7.93 (m, 6H),7.51-7.36 (m, 9H), 5.69-5.64 (m, 1H), 5.60-5.56 (m, 2H), 4.80 (m, 1H),4.57 (dd, 1H, J=2.7, 12.0 Hz), 4.52 (dd, 1H, J=12.0, 5.5 Hz), 2.72-2.54(m, 3H), 2.41-2.35 (m, 1H), 1.30 (t, 3H, J=7.4 Hz); ¹³C NMR (CDCl₃):165.2, 164.6, 164.5, 132.3, 132.1, 132.0, 128.8, 128.7, 128.6, 128.4,128.1, 127.4, 127.3, 78.5, 69.4, 67.3, 62.4, 34.4, 23.8. MS: calcd forC₂₉H₂₈O₇SNa 543.1, found m/z 543.1 (M+Na). α isomer: [α]_(D)=−46° (c=1,CHCl₃) IR: 2961, 2923, 1732, 1717, 1269, 1108, 1099, 708, 685 cm⁻¹. ¹HNMR (CDCl₃) 8.12-7.93 (m, 6H), 7.54-7.37 (m, 9H), 5.56 (t, 1H, J=9.7Hz), 5.46 (m, 1H), 4.87 (dd, 1H, J=11.8, 1.7 Hz), 4.60 (dd, 1H, J=3.1,12.0 Hz), 4.48 (dd, 1H, J=5.9, 12.0 Hz), 4.06 (m, 1H), 2.83-2.68 (m,2H), 2.65-2.64 (m, 1H), 2.08 (m, 1H), 1.32 (t, 3H, J=7.4 Hz); ¹³C NMR(CDCl₃) 166.6, 166.3, 165.9, 134.1, 133.8, 133.7, 133.4, 130.6, 130.1,130.0, 129.7, 129.5, 128.9, 128.7, 80.3, 77.1, 73.0, 70.5, 64.3, 37.1,25.5, 15.5. MS: calcd for C₂₉H₂₈O₇SNa 543.1, found: m/z 543.0 (M+Na).

[0183] Ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-gulopyranoside (30).

[0184] Compound 29 (0.75 g, 1.07 mmol) gave 0.65 g of the desiredcompound (94%). ¹H NMR (CDCl₃) 8.18-7.87 (m, 8H), 7.54-7.27 (m, 12H),5.95 (t, J=3.5 Hz, 1H), 5.67 (dd, J=3.3, 10.3 Hz, 1H), 5.61 (m, 1H),5.27 (d, J=10.3 Hz, 1H), 4.64 (m, 2H), 4.50 (dd, J=3.8, 9.5 Hz, 1H),2.84 (m, 2H), 1.34 (t, J=7.4 Hz, 3H); MS: calcd for C₃₆H₃₂O₉SNa 663.2,found m/z 663.1 (M+Na).

[0185] Ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-allopyranoside (35).

[0186] Compound 34 (0.97 g, 1.38 mmol) gave 0.85 g desired product(95%). [α]_(D)=12.7° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.07-7.32 (m, 20H),6.26 (t, J=2.8 Hz, 1H), 5.56 (dd, J=2.8, 10.1 Hz, 1H), 5.51 (dd, J=2.9,10.1 Hz, 1H), 4.71 (dd, J=2.5, 12.0 Hz, 1H), 4.56 (m, 1H), 4.47 (dd,J=5.3, 12.0 Hz, 1H), 2.80 (m, 2H), 1.29 (t, J=7.5 Hz, 3H); ¹³C NMR(CDCl₃) 171.6, 166.6, 165.7, 165.2, 134.0, 133.9, 133.8, 133.6, 130.5,130.3, 130.2, 130.1, 130.0, 129.9, 129.6, 129.3, 129.2, 128.9, 128.8,81.3, 73.7, 69.7, 68.9, 68.0, 63.9, 24.3, 15.5; MS: calcd forC₃₆H₃₂O₉SNa 663.1, found m/z 663.0 (M+Na).

[0187] General Strategy for O-Benzoyl to O-Benzyl Conversion.

[0188] O-Benzoyl may be converted to O-Benzyl according to the followingmethod or other methods known to those skilled in the art. Protectedethyl 1-thio-β-D-hexopyranoside are dissolved in dry MeOH and toluene towhich a sodium methoxide solution is added. The mixture is then stirred,preferably for about 1½ to 2½ hrs at room temperature and optionallyneutralized. The organics are preferably concentrated and thecorresponding unprotected 1-ethylthio-β-D-glucopyranoside purified, andthen dissolved. NaH is then added and the reaction is stirred for about1½ to 2½ hrs at room temperature followed by the addition of benzylbromide and stirring, preferably overnight. The mixture may then bediluted, washed with H₂O, brine, and organics, dried, concentrated, andpurified to give the purified product.

[0189] Other conditions, reagents, solutions and the like of the presentmethod, or other methods known to those skilled in the art may be usedin accordance with the present invention.

[0190] In a typical reaction, 1.4 mmol of protected ethyl1-thio-β-D-hexopyranoside was dissolved in 10 mL dry MeOH and 3 mLtoluene to which 0.25 mL of a sodium methoxide solution (25% NaOMe inmethanol) was added. The mixture was stirred for 2 hr at roomtemperature and neutralized with 1N acetic acid. The organics wereconcentrated and the corresponding unprotected1-ethylthio-β-D-glucopyranoside purified by silica gel chromatography(10:1 hexane/EtOAc) which was then dissolved in 10 mL dry DMF and 323 mg65% NaH (8.0 mmol) was added. The reaction was stirred for 2 hr at roomtemperature followed by the addition of 1 mL benzyl bromide (8.3 mmol)and continued stirring overnight. The mixture was then diluted with 150mL EtOAc, washed with H₂O (30 mL), brine (30 mL) and the organics driedover Na₂SO₄, concentrated and purified by silica gel chromatography(10:1 hexane/EtOAc) to give the purified product.

[0191] Ethyl 2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (10).

[0192] Compound 9 (0.7 g, 1.35 mmol) gave 480 mg (75%) of purifiedproduct. [α]_(D)=5.8° (c=1.0, CHCl₃); ¹H NMR (CDCl₃) 7.40-7.29 (m, 15H),4.95-4.85 (m, 4H), 4.77 (d, 1H, J=10.2 Hz), 4.65 (d, 1H, J=10.5 Hz),4.48 (d, 1H, J=9.8 Hz), 3.66 (t, 1H, J=8.9 Hz), 3.46-3.38 (m, 2H), 3.23(t, 1H, J=9.2 Hz), 2.84-2.70 (m, 2H), 1.35-1.27 (m, 2H); ¹³C NMR(CDCl₃): 138.9, 138.5, 138.4, 128.9, 128.8, 128.7, 128.4, 128.3, 128.2,128.1, 86.8, 85.2, 83.8, 82.5, 76.2, 75.9, 75.8, 25.4, 18.5, 15.5. MS:calcd for C₂₉H₃₄O₄SNa 501.2, found m/z 501.1 (M+Na).

[0193] Ethyl 2,3,6-tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside (18).

[0194] Compound 17 (0.85 g, 1.63 mmol) gave 675 mg (86%) purifiedproduct. [α]_(D)=40° (c=1, CHCl₃), ¹H NMR (CDCl₃) 7.45-7.31 (m, 1SH),4.92 (d, 1H, J=10.3 Hz), 4.86 (d, 1H, J=10.3 Hz), 4.74 (d, 1H, J=11.7Hz), 4.69 (d, 1H, J=11.7 Hz), 4.62 (d, 1H, J=12.0 Hz), 4.58 (d, 1H,J=12.0 Hz), 4.49 (d, 1H, J=9.7 Hz), 3.69-3.62 (m, 3H), 3.50 (m, 1H),3.36 (dd, 1H, J=8.7, 9.4 Hz), 2.82-2.75 (m, 2H), 2.23 (m, 1H), 1.54 (m,1H), 1.35 (t, 3H, J=7.5 Hz); ¹³C NMR (CDCl₃) 138.8, 138.7, 138.5, 128.8,128.7, 128.6, 128.2, 128.1, 128.0, 85.5, 82.3, 80.6, 76.0, 75.4, 73.9,72.9, 72.3, 34.4, 25.2, 15.6 MS: calcd for C₂₉H₃₄O₄SNa 501.2, found m/z501.0 (M+Na).

[0195] Ethyl 2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (26).

[0196] Compound 25 (608 mg, 1.17 mmol) gave 364 mg substantially pureproduct (65%). [α]_(D)=−11.8° (c=1, CHCl₃); ¹H NMR (CDCl₃) 7.44-7.14 (m,15H), 4.74 (d, 1H, J=11.6 Hz), 4.66-4.56 (m, 4H), 4.50 (d, 1H, J=9.4Hz), 4.45 (d, 1H, J=11.4 Hz), 3.83 (d, 1H, J=10.7 Hz), 3.69 (dd, 1H,J=4.4, 10.7 Hz), 3.49 (m, 2H), 3.35 (m, 1H), 2.79 (m, 2H), 2.69 (m, H),1.54 (m, 1H), 1.35 (t, 3H, J=7.3 Hz); ¹³C NMR (CDCl₃): 138.8, 138.4,135.8, 128.9, 128.7, 128.4, 128.2, 128.1, 127.4, 86.9, 81.3, 75.6, 73.8,73.3, 72.5, 71.6, 69.8, 45.3, 36.7, 25.1, 20.3, 15.5; MS: calcd forC₂₉H₃₄O₄SNa 501.2, found m/z 501.0 (M+Na).

[0197] Ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-gulopyranoside (31).

[0198] Compound 30 (0.6 g, 0.94 mmol) gave 330 mg substantially pureproduct (60%). ¹H NMR (CDCl₃) 7.32-7.17 (m, 20H), 4.94 (d, J=9.8 Hz,1H), 4.55 (m, 2H), 4.40 (m, 4H), 4.22 (m, 2H), 4.00 (t, J=6.4 Hz, 1H),3.60-3.42 (m, 5H), 2.66 (m, 2H), 1.22 (t, J=7.4 Hz, 3H); MS: calcd forC₃₆H₄₀O₅SNa 607.2, found m/z 607.0 (M+Na).

[0199] Ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-allopyranoside (36).

[0200] Compound 35 (0.85 g, 1.33 mmol) gave 496 mg substantially pureproduct (64%). ¹H NMR (CDCl₃) 7.4-7.22 (m, 20H), 5.05 (d, J=9.7 Hz, 1H),4.86 (d, J=11.8 Hz, 1H), 4.80 (d, J=11.8 Hz, 1H), 4.69-4.40 (m, 6H),4.13 (m, 1H), 4.03 (dd, J=3.1, 9.7 Hz, 1H), 3.47 (dd, J=2.3, 9.8 Hz,1H), 3.29 (dd, J=2.3, 9.8 Hz, 1H), 2.75 (m, 2H), 1.32 (t, J=7.5 Hz, 3H);¹³C NMR (CDCl₃) 139.4, 138.9, 138.3, 138.2, 128.9, 128.8, 128.7, 128.6,128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 82.0, 79.2, 76.1, 75.4,74.7, 73.9, 73.8, 72.8, 71.9, 69.9, 25.1, 15.6; MS: calcd forC₃₆H₄₀O₅SNa 607.2, found m/z 607.0 (M+Na).

[0201] General Phosphorylation Strategy (Method A: via Ethyl1-thio-β-D-hexopyranoside).

[0202] Phosphorylation may take place in accordance with the presentinvention by the following reaction, which involves ethyl1-thio-β-D-hexopyranoside. The ethyl 1-thio-β-D-hexopyranoside may beethyl 1-thio-β-D-hexopyranoside prepared according to the methodsdescribed herein or ethyl 1-thio-β-D-hexopyranoside prepared by othermethods.

[0203] According to this method, protected ethyl1-thio-β-D-hexopyranoside and dibenzyl phosphate are co-evaporated,preferably two times from dry toluene and further dried under highvacuum overnight to which N-iodosuccinamide and dry molecular sieves arepreferably added. The mixture is then dissolved, preferably in dryCH₂Cl₂, cooled to about −40° C. to about −20° C., preferably about −30°C. and trifluoromethane-sulfonic acid is added. The reaction mixture issubstantially maintained at the cooled temperature for about 20 to about40 minutes, preferably about 30 min with stirring. Preferably, themixture is then diluted, and washed with saturated Na₂S₂O₃ and/orsaturated NaHCO₃, H₂O, and brine. The organics are then preferablydried, filtered, concentrated and purified to give the desired product.

[0204] Other conditions, steps, reagents, solutions and the like of thepresent method may be used in accordance with the present invention.

[0205] In a typical reaction, 0.84 mmol protected ethyl1-thio-β-D-hexopyranoside and 1.44 mmol dibenzyl phosphate wereco-evaporated two times from dry toluene and further dried under highvacuum overnight to which 1.24 mmol of N-iodosuccinamide and 300 mg drymolecular sieves were added. The mixture was then dissolved in 10 mL dryCH₂Cl₂, cooled to −30° C. and 25 μl trifluoromethanesulfonic acid (0.28mmol) was added. The reaction mixture was maintained at −30° C. for 30min with stirring and then diluted with 100 mL EtOAc, washed withsaturated Na₂S₂O₃ (20 mL) and saturated NaHCO₃ (20 mL), H₂O (20 mL), andbrine (20 mL). The organics were dried over Na₂SO₄, filtered,concentrated and purified by chromatography on silica gel (3:1hexane/EtOAc) to give the desired product.

[0206] Dibenzyl-(2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl)Phosphate (11).

[0207] Compound 10 (400 mg, 0.84 mmol) gave 0.44 mg (76%) of the desiredproduct. [α]_(D)=22.8° (c=1, CHCl₃); ¹H NMR (CDCl₃) 7.38-7.28 (m, 25H),5.93 (dd, 1H, J=3.2, 6.6 Hz), 5.30 (m, 4H), 5.18 (m, 3H), 5.09 (m, 2H),4.67 (m, 2H), 3.94 (m, 1H), 3.64 (m, 1H), 3.18 (m, 1H), 1.21 (d, 3H,J=6.2 Hz); ¹³C NMR (CDCl₃) 138.9, 138.5, 138.0, 128.9, 128.8, 128.7,128.4, 128.3, 95.8, 95.7, 94.0, 76.0, 75.7, 73.6, 69.7, 17.3; ³¹P NMR(CDCl₃) 2.58; MS: calcd for C₄₁H₄₃O₈PNa 717.2, found m/z 717.3 (M+Na).

[0208] Dibenzyl-(2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl)Phosphate (19).

[0209] Compound 18 (512 mg, 1.07 mmol) gave 0.565 g (76%) substantiallypure product. [α]_(D)=28.2° (c=1, CHCl₃); ¹H NMR (CDCl₃) 7.29-7.10 (m,25H), 5.91(dd, 1H, J=3.2, 6.6 Hz), 4.72-4.57 (m, 4H), 4.41 (m, 2H), 4.02(m, 1H), 3.81 (m, 1H), 3.48 (m, 1H), 3.34 (m, 2H), 2.04-2.00 (m, 1H),1.60-1.48 (m, 1H); ¹³C NMR (CDCl₃) 138.6, 138.4, 138.2, 137.8, 137.7,128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 97.3,91.9, 83.6, 80.2, 78.1, 74.9, 73.3, 73.0, 72.2, 72.0, 71.9, 70.6, 66.7,52.7, 33.4; ³¹P NMR (CDCl₃) 1.25; MS: calcd for C₄₁H₄₃O₈PNa 717.2, foundm/z 717.2 (M+Na).

[0210] Dibenzyl-(2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)Phosphate (27).

[0211] Compound 26 (270 mg, 0.56 mmol) gave 0.31 g substantially pureproduct (79%, α/β=2:1). ¹H NMR (CDCl₃) 7.32-7.21 (m, 25H), 5.96 (dd, 1H,J=2.8, 6.6 Hz), 5.06 (m, 4H), 4.66 (d, 1H, J=11.7 Hz), 4.56 (m, 3H),4.42 (d, 1H, J=12.0 Hz), 4.38 (d, 1H, J=11.3 Hz), 3.82 (m, 1H), 3.66 (m,3H), 3.49 (m, 1H), 2.54 (m, 0.5H), 2.40 (m, 1H), 1.85 (m, 1H), 1.56 (m,0.5H); ³¹P NMR (CDCl₃) 0.54, 0.17; MS: calcd for C₄₁H₄₃O₈PNa 717.2,found m/z 717.2 (M+Na).

[0212] Dibenzyl-(3,4,6-tri-O-benzoyl-2-deoxy-α-D-glucopyranosyl)Phosphate (41).

[0213] Compound 40 (460 mg, 0.88 mmol) gave 0.49 g of substantially pureproduct (75%) after silica gel chromatography (3:1-2:1 hexane/EtOAc.[α]_(D)=19° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.05-7.94 (m, 6H), 7.53-7.51(m, 3H), 7.41-7.34 (m, 16H), 5.96 (dd, 1H, J=1.6, 7.2 Hz), 5.68 (m, 2H),5.16 (m, 4H), 4.51-4.43 (m, 2H), 4.35 (dd, 1H, J=3.1, 12.0 Hz), 2.56 (m,1H), 2.04 (m, 1H); ¹³C NMR (CDCl₃) 166.0, 165.6, 165.3, 135.5, 135.4,133.3, 133.2, 133.0, 129.8, 129.7, 129.6, 129.3, 128.9, 128.6, 128.4,128.3, 128.1, 127.9, 95.9, 70.1, 69.5, 69.2, 68.9, 62.6; ³¹P NMR (CDCl₃)0.32; MS: calcd for C₄₁H₃₇O₁₁PNa 759.1, found: m/z 759.1 (M+Na).

[0214] Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-gulopyranosyl) Phosphate(32).

[0215] Compound 31 (120 mg, 0.21 mmol) gave 50 mg of the desiredcompound (30%) and 38 mg of the β isomer (23%). α isomer: ¹H NMR (CDCl₃)7.30-6.90 (m, 30H), 5.95 (dd, J=3.7, 7.5 Hz, 1H), 4.97 (m, 5H), 4.64 (m,2H), 4.49-4.30 (m, 8H), 3.80 (m, 2H), 3.60 (d, J=3.4 Hz, 1H), 3.44 (m,2H); ³¹P NMR (CDCl₃) 0.8; MS: calcd for C₄₈H₄₉O₉PNa 823.3, found m/z823.3 (M+Na). β isomer: ¹H NMR (CDCl₃) 7.25-7.15 (m, 30H), 5.61 (t,J=7.2 Hz, 1H), 5.05-4.99 (m, 4H), 4.60 (d, J=12 Hz, 1H), 4.42-4.34 (m,4H), 4.26 (d, J=6.2 Hz, 1H), 4.17 (t, J=6.3 Hz, 1H), 3.65 (m, 2H), 3.48(m, 2H), 3.48 (m, 2H), 3.43 (dd, J=1.3, 13.5 Hz, 1H); ³¹P NMR (CDCl₃)−1.1; MS: calcd for C₄₈H₄₉O₉PNa 823.3, found m/z 823.3 (M+Na).

[0216] Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-allopyranosyl) Phosphate(37).

[0217] Compound 36 (169 mg, 0.29 mmol) gave 70 mg the desired compound(30%) and 64 mg of the β isomer (28%). α isomer: ¹H NMR (CDCl₃)7.34-7.13 (m, 30H), 6.04 (dd, J=3.6, 7.1 Hz, 1H), 5.11-4.92 (m, 4H),4.89 (d, J=12.0 Hz, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.75 (d, J=11.8 Hz,1H), 4.59-4.37 (m, 6H), 4.23 (m, 1H), 3.73 (dd, J=3.0, 10.0 Hz, 1H),3.66 (dd, J=2.5, 10.0 Hz, 1H), 3.54 (m, 2H); ¹³C NMR (CDCl₃) 139.4,138.4, 138.3, 137.9, 136.5, 136.4, 128.9, 128.8, 128.7, 128.7, 128.5,128.4, 128.3, 128.2, 128.1, 128.0, 127.6, 95.1, 76.2, 74.4, 73.9, 73.3,71.8, 69.6, 69.2, 68.7, 68.6; ³¹P NMR (CDCl₃) 0.27; MS: calcd forC₄₈H₄₉O₉PNa 823.3, found m/z 823.3 (M+Na). β isomer: ¹H NMR (CDCl₃)7.37-7.07 (m, 30H), 5.63 (t, J=7.7 Hz, 1H), 5.00 (m, 4H), 4.79 (d,J=11.9 Hz, 1H), 4.72 (d, J=11.9 Hz, 1H), 4.63 (d, J=11.9 Hz, 1H), 4.51(d, J=11.9 Hz, 1H), 4.46 (d, J=12.1 Hz, 1H), 4.36 (m, 3H), 4.09 (dd,J=1.4, 9.7 Hz, 1H), 4.02 (s, 1H), 3.64 (dd, J=3.7, 11.0 Hz, 1H), 3.58(dd, J=1.5, 11.0 Hz, 1H), 3.51 (dd, J=2.3, 9.8 Hz, 1H), 3.32 (dd, J=2.3,7.9 Hz, 1H; ³¹P NMR (CDCl₃) 0.76; MS: calcd for C₄₈H₄₉O₉PNa 823.3, foundm/z 823.3 (M+Na).

[0218] General Phosphorylation Strategy (Method B: via Glycosyl Halide).

[0219] According to another embodiment, phosphorylation may take placein accordance with the present invention by the following reaction,which involves glycosyl halide. The glycosyl halide may be glycosylhalide prepared according to the methods described herein or glycosylhalide prepared by other methods.

[0220] According to this method, protected D-hexose is dissolved inacetic acid to which HBr in acetic acid was added dropwise at about 0°C. The reaction is allowed to warm to room temperature and stirred forabout 1½ to about 2½ hrs. The mixture is then diluted with cold CHCl₃,washed successively with cold saturated NaHCO₃ solution, H₂O and brine,and the organics were dried over anhydrous Na₂SO₄ and concentrated. Thecrude protected-α-D-pyranosyl bromide may be used directly withoutfurther purification. A mixture of dibenzyl phosphate, silver triflate,2,4,6-collidine and activated 4 Å molecular sieves in dry CH₂Cl₂ isstirred at room temperature in the absence of light for about 1 hr. Themixture was then cooled to about −30° C. to about −50° C., preferablyabout −40° C., to which a solution of the crude protected-α-D-pyranosylbromide in dry CH₂Cl₂ is added in dropwise fashion. The reaction mixtureis kept at substantially the same cool temperature for about 1½ to about2½ M hrs, allowed to warm to room temperature and stirred, preferablyovernight. The corresponding filtrate is preferably diluted with CH₂Cl₂,washed with saturated CuSO₄, H₂O, and brine, and the organics aree driedover anhydrous Na₂SO₄ and concentrated. Purification yieldssubstantially pure product.

[0221] Other conditions, steps, reagents, solutions and the like of thepresent method may be used in accordance with the present invention.

[0222] Suitably protected D-hexose (0.64 mmol) was dissolved in 5 mLacetic acid to which 5 mL 33% HBr in acetic acid was added dropwise at0° C. The reaction was allowed to warm to room temperature and stirredfor 2 hr. The mixture was then diluted with 100 mL cold CHCl₃, washedsuccessively with cold saturated NaHCO₃ solution (3×30 mL), H₂O (30 mL)and brine (20 mL), and the organics were dried over anhydrous Na₂SO₄ andconcentrated. The crude protected-α-D-pyranosyl bromide was useddirectly without further purification. A mixture of dibenzyl phosphate(1.80 mmol), silver triflate (1.80 mmol), 2,4,6-collidine (3.0 mmol) and0.5 g activated 4 Å molecular sieves in 10 mL dry CH₂Cl₂ was stirred atroom temperature under argon atmosphere in the absence of light for 1hr. The mixture was then cooled to −40° C. to which a solution of thecrude protected-α-D-pyranosyl bromide in 10 mL dry CH₂Cl₂ was added indropwise fashion. The reaction mixture was kept at −40° C. for 2 hr,allowed to warm to room temperature and stirred overnight. Thecorresponding filtrate was diluted with 100 mL CH₂Cl₂, washed withsaturated CuSO₄ (2×20 mL), H₂O (20 mL) and brine (20 mL), and theorganics were dried over anhydrous Na₂SO₄ and concentrated. Purificationby silica gel chromatography (1:1 hexanes/EtOAc) gave substantially pureproduct.

[0223] Dibenzyl-(2,3,4,6-tetra-O-benzoyl-α-D-altropyranosyl) Phosphate(45).

[0224] Perbenzoylated D-altrose (44), (0.675 g, 0.96 mmol) gave 0.58 gsubstantially pure product (70% overall). [α]_(D)=40° (c=1, CHCl₃); ¹HNMR (CDCl₃) 8.05 (m, 6H), 7.82 (dd, J=1.2, 7.2 Hz, 2H), 7.53-7.22 (m,22H), 5.87 (m, 3H), 5.38 (d, J=3.1 Hz, 1H), 5.04 (m, 4H), 4.90 (dd,J=3.0, 10.0 Hz, 1H), 4.58 (dd, J=2.4, 12.3 Hz, 1H), 4.38 (dd, J=3.9,12.3 Hz, 1H); ¹³C NMR (CDCl₃) 166.4, 165.4, 165.3, 164.8, 135.7, 134.3,133.9, 133.5, 130.5, 130.4, 130.3, 130.1, 129.6, 129.3, 129.1, 129.0,128.9, 128.8, 128.4, 128.3, 70.2, 70.1, 70.0, 69.5, 69.4, 67.1, 66.9,65.5, 63.0; ³¹P NMR (CDCl₃) −0.02; MS: calcd for C₄₈H₄₁O₁₃NaP 879.2,found m/z 879.2 (M+Na).

[0225] Dibenzyl-(2,3,4,6-tetra-O-benzoyl-α-D-idopyranosyl) Phosphate(49)

[0226] Perbenzoylated D-idose (48), (0.32 g, 0.46 mmol) gave 270 mgsubstantially pure product. (69% overall). [α]_(D)=11.4° (c=1, CHCl₃);¹H NMR (CDCl₃) 8.11-7.88 (m, 8H), 7.40-7.19 (m, 22H), 6.0 (d, J=6.5 Hz,1H), 5.68 (m, 1H), 5.46 (m, 1H), 5.22 (m, 1H), 5.07-5.01 (m, 5H), 4.60(dd, J=7.0, 11.5 Hz, 1H), 4.53 (dd, J=5.8, 11.5 Hz, 1H); ¹³C NMR (CDCl₃)166.0, 165.1, 164.7, 164.3, 135.2, 133.6, 133.5, 133.1, 130.1, 130.0,129.9, 129.7, 129.6, 129.4, 128.9, 128.6, 128.5, 128.4, 128.3, 128.2,127.9, 127.8, 94.9, 69.7, 69.5, 65.9, 65.7, 62.6; ³¹P NMR (CDCl₃) 0.1;MS: calcd for C₄₈H₄₁O₁₃NaP 879.2, found m/z 879.1 (M+Na).

[0227] Dibenzyl-(2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl) Phosphate(53).

[0228] Peracylated D-talose (52), (0.248 g, 0.636 mmol) gave 0.436 gsubstantially pure product (52% overall) [α]_(D)=40° (c=1, CHCl₃); ¹HNMR (CDCl₃) 7.37 (m, 10H), 5.68 (dd, J=1.3, 6.5 Hz, 1H), 5.31 (m, 1H),5.20 (t, J=3.7 Hz, 1H), 5.11 (m, 4H), 5.04 (d, J=3.0 Hz, 1H), 4.30 (dd,J=1.3, 6.8 Hz, 1H), 4.11 (dd, J=11.3, 6.7 Hz, 1H), 3.99 (dd, J=11.3, 6.7Hz, 1H), 2.12 (s, 3H), 2.11 (s, 3H), 1.99 (s, 3H), 1.92 (s, 3H); ¹³C NMR(CDCl₃) 170.7, 170.4, 169.9, 169.8, 135.7, 135.6, 135.5, 129.2, 129.1,129.0, 128.5, 128.4, 96.3, 70.3, 68.9, 67.0, 65.6, 64.9, 61.7, 21.1,21.0, 20.9; ³¹P NMR (CDCl₃) −0.18; HRMS (FAB) calcd for C₂₈H₃₄O₁₃P609.1737, found m/z 609.1747 (M+H).

[0229] General Strategy for Final Deprotection and Conversion to theSodium Salt.

[0230] Final Deprotection and Conversion to sodium salt may take placein accordance with the present invention by the following reaction.

[0231] According to this method, protected α-D-pyranosyl phosphate isdissolved in MEOH, NaHCO₃ solution and 10% Pd/C are added. The mixtureis stirred overnight at room temperature under hydrogen atmosphere afterwhich the catalyst is removed, preferably by filtration and the filtrateconcentrated. The aqueous layer is preferably extracted, and thenpartitioned and submitted to an anion exchange column eluted with water,0.1M NH₄HCO₃, 0.2M NH₄HCO₃ and 0.3M NH₄HCO₃. The product eluted with 0.2M NH₄HCO₃ and these fractions are pooled and co-evaporated with ethanol,preferably several times to remove excess NH₄HCO₃. The obtained sugarphosphate ammonium salt is subsequently dissolved in water and appliedto a cation-exchange column (Na⁺ type) eluted with mL water. The productcontaining fractions are collected and lyophilized to give the desiredproduct as the sodium salt.

[0232] Other conditions, steps, reagents, solutions and the like of thepresent method may be used in accordance with the present invention.

[0233] In a typical reaction, the protected α-D-pyranosyl phosphate (0.5mmol) was dissolved in 15 mL MeOH, 1.5 mL 1N NaHCO₃ solution and 150 mg10% Pd/C were added. The mixture was stirred overnight at roomtemperature under hydrogen atmosphere after which the catalyst wasremoved by filtration and the filtrate concentrated to approximately a10 mL volume. The aqueous layer was extracted with 10 mL of EtOAc, andthen partitioned and submitted to an anion exchange column (Dowex 1×8,1.2×12 cm) eluted with 100 mL water, 100 mL 0.1 M NH₄HCO₃, 100 mL 0.2 MNH₄HCO₃ and 100 mL 0.3 M NH₄HCO₃. The product eluted with 0.2M NH₄HCO₃and these fractions were pooled and co-evaporated with ethanol severaltimes to remove excess NH₄HCO₃. The obtained sugar phosphate ammoniumsalt was subsequently dissolved in 5 mL water and applied to an AG-X8cation-exchange column (Na⁺ type) eluted with 100 mL water. The productcontaining fractions were collected and lyophilized to give the desiredproduct as the sodium salt.

[0234] Disodium 6-deoxy-α-D-glucopyranosyl Phosphate (12).

[0235] Compound 11 (350 mg, 0.5 mmol) gave 85 mg (58%) of the desiredsodium salt. ¹H NMR (D₂O) 5.37 (dd, 1H, J=3.4, 7.2 Hz), 3.98 (m, 1H),3.70 (t, 1H, J=9.5 Hz), 3.45 (m, 1H), 3.09 (t, 1H, J=9.5 Hz), 1.24 (d,1H, J=6.2 Hz); ¹³C NMR (D₂O) 93.74, 75.78, 73.3, 72.9, 68.2, 17.2; ³¹PNMR (D₂O) 3.02; HRMS (FAB): calcd for C₆H₁₂O₈P 243.0269, found m/z243.0277 (M+H).

[0236] Disodium 4-deoxy-α-D-glucopyranosyl Phosphate (20).

[0237] Compound 19 (342 mg, 0.5 mmol) gave 78 mg of the title compound(55%). ¹H NMR (D₂O) 5.49 (dd, 1H, J=3.4, 7.32 Hz), 4.16 (m, 1H), 3.99(m, 1H), 3.65 (dd, 1H, J=3.2, 12.0 Hz), 3.55 (dd, 1H, J=6.0, 12.0 Hz),3.41 (m, 1H), 1.99-1.95 (m, 1H), 1.44 (m, 1H); ¹³C NMR (D₂O) 95.1, 73.8,69.5, 67.4, 64.0, 34.3; ³¹P NMR (D₂O) 1.52; HRMS (FAB): calcd forC₆H₁₂O₈P 243.0269, found m/z 243.0260 (M+H).

[0238] Disodium 3-deoxy-α-D-glucopyranosyl Phosphate (28).

[0239] Compound 27 (270 mg, 0.39 mmol) gave 65 mg title compound (58%)as a 2:1 α/β mixture. ¹H NMR (D₂O) 5.33 (dd, 1H, J=3.2, 7.3 Hz),3.92-3.50 (m, 5H), 3.46 (m, 1H), 2.33 (m, 0.43H), 2.12 (m, 1H), 1.81 (m,1H), 1.54 (m, 0.43H); ³¹P NMR (D₂O) 3.39, 3.12; HRMS (FAB): calcd forC₆H₁₂O₈P 243.0269, found m/z 243.0267 (M+H).

[0240] Disodium 2-deoxy-α-D-glucopyranosyl Phosphate (43).

[0241] Debenzylation of 41 (329 mg, 0.447 mmol) was accomplished usingthe general strategy described above. After the filtrate wasconcentrated to approximately a 10 mL volume, the solution was cooled to0° C. and 1.5 mL 1N NaOH solution was added in dropwise manner. Themixture was then stirred at room temperature for 4 hr and subsequentlyneutralized with 1.0 N acetic acid. The final work-up was accomplishedas described in the general strategy to give 69 mg title compound (53%).¹H NMR (D₂O) 5.53 (m, 1H), 4.01 (m, 1H), 3.88-3.84 (m, 3H), 3.72 (dd,1H, J=6.2, 12.7 Hz), 3.31 (t, 1H, J=9.4 Hz), 2.19 (dd, 1H, J=5.0, 12.9Hz), 1.66 (m, H); ³¹P NMR (D₂O) 2.68; HRMS (FAB): calcd for C₆H₁₂O₈P243.0269, found m/z 243.0268 (M+H).

[0242] Disodium α-D-gulopyranosyl Phosphate (33).

[0243] Compound 32 (35 mg, 0.044 mmol) gave 7.1 mg of the title compound(55%). ¹H NMR (D₂O) 5.15 (dd, J=3.0, 7,7 Hz, 1H), 4.04 (m, 2H), 3.79 (m,2H), 3.64 (m, 2H); ¹³C NMR (D₂O) 96.1, 75.3, 71.6, 70.2, 70.0, 62.2; ³¹PNMR (D₂O) 2.9; HRMS (FAB): calcd for C₆H₁₂O₉P 259.0218, found m/z259.0231 (M+H).

[0244] Disodium α-D-allopyranosyl Phosphate (38).

[0245] Compound 37 (63 mg, 0.079 mmol) gave 18 mg substantially pureproduct (77%). ¹H NMR (D₂O) 5.44 (dd, J=3.5, 7.5 Hz, 1H), 4.14 (m, 1H),4.00 (m, 1H), 3.90 (dd, J=1.9, 12.3 Hz, 1H), 3.76 (m, 2H), 3.65 (dd,J=3.0, 10.4 Hz, 1H); ¹³C NMR (D₂O) 95.8, 75.1, 72.6, 71.8, 68.1, 62.6.;³¹P NMR (D₂O) 2.39; HRMS (FAB): calcd for C ₆H₁₂O₉P 259.0218, found m/z259.0217 (M+H).

[0246] Disodium α-D-altropyranosyl Phosphate (47).

[0247] Using the strategy described for 43, compound 45 (260 mg, 0.3mmol) gave 62 mg of the desired sodium salt (67% overall). ¹H NMR (D₂O)5.29 (d, J=8.4 Hz, 1H), 4.14 (m, 1H), 3.98 (m, 1H), 3.94 (t, J=3.5 Hz,1H), 3.90 (dd, J=2.4, 12.3 Hz, 1H), 3.82 (dd, J=3.5, 12.4 Hz, 1H), 3.77(dd, J=6.5, 12.3 Hz, 1H); ¹³C NMR (D₂O) 94.9, 70.6, 70.5, 70.0, 64.8,61.4; ³¹P NMR (D₂O) 2.05; HRMS (FAB): calcd for C₆H_(12 O) ₉P 259.0218,found m/z 259.0211 (M+H).

[0248] Disodium α-D-idopyranosyl Phosphate (51).

[0249] Using the strategy described for 43, compound 49 (213 mg, 0.25mmol) gave 61 mg of the title compound (62% overall). ¹H NMR (D₂O) 5.14(dd, J=3.5, 7.7 Hz, 1H), 4.24 (m, 1H), 3.85 (dd, J=8.9, 12.3 Hz, 1H),3.75 (m, 2H), 3.60 (t, J=5.0 Hz, 1H), 3.32 (m, 1H); ¹³C NMR (D₂O) 99.1,75.8, 75.5, 74.1, 63.9, 53.2; ³¹P NMR (CDCl₃) 2.98; HRMS (FAB): calcdfor for C₆H₁₂O₉P 259.0218, found m/z 259.0208 (M+H).

[0250] Disodium α-D-talopyranosyl Phosphate (55).

[0251] Using the strategy described for 43, compound 53 (436 mg, 0.72mmol) gave 157 mg of the title compound (72%). ¹H NMR (D₂O) 5.48 (d,J=8.2 Hz, 1H), 4.11 (m, 1H), 3.98 (t, J=3.2, 1H), 3.92 (m, 1H), 3.88 (m,1H), 3.82 (dd, J=11.1, 7.7 Hz, 1H), 3.75 (dd, J=11.7, 4.4 Hz, 1H), 3.19(q, J=7.3 Hz, 10H), 1.28 (t, J=7.4, 15H); ¹³C NMR (D₂O): 94.1, 70.2,68.8, 67.6, 62.8, 59.6; ³¹P NMR (D₂O) 0.52; HRMS (FAB): calcd forC₆H₁₂O₉P 259.0218, found m/z 259.0209 (M+H).

[0252] The following compounds were prepared, preferably according tothe methods described herein.

[0253] (58) Thymidine 5′-(α-D-glucopyranosyl diphosphate). HRMS (FAB)calc for C₁₆H₂₅O₁₆N₂P₂ 563.0705; found m/z 563.0679 (M+H).

[0254] (59) Uridine 5′-(α-D-glucopyranosyl diphosphate). HRMS (FAB):calc for C₁₄H₂₃O₁₇N₂P₂ 565.0507; found m/z 565.0472 (M+H).

[0255] (60) Thymidine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB) calc for C₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0714 (M+H).

[0256] (61) Uridine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc C₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0510 (M+H).

[0257] (62) Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB) calc for C₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0720 (M+H).

[0258] (63) Uridine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc C₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0485 (M+H).

[0259] (64) Thymidine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB) calc for C₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0693 (M+H).

[0260] (65) Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc C₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0500 (M+H).

[0261] (66) Thymidine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB) calc for C₁₆H₂₅O₁₅N₂P₂ 547.0704; found m/z 547.0730 (M+H).

[0262] (67) Uridine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate). HRMS(FAB): calc C₁₄H₂₃O₁₆N₂P₂ 549.0506; found m/z 549.0492 (M+H).

[0263] (68) Thymidine 5′-(α-D-mannopyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.0701 (M+H).

[0264] (69) Uridine 5′-(α-D-mannopyranosyl diphosphate). HRMS (FAB):calc 565.0507; found m/z 565.0503 (M+H).

[0265] (70) Thymidine 5′-(α-D-galactopyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.0710 (M+H).

[0266] (71) Uridine 5′-(α-D-galactopyranosyl diphosphate). HRMS (FAB):calc 565.0507; found m/z 565.0508 (M+H).

[0267] (72) Thymidine 5′-(α-D-allopyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.0715 (M+H).

[0268] (73) Uridine 5′-(α-D-allopyranosyl diphosphate). HRMS (FAB): calc565.0507; found m/z 565.0507 (M+H).

[0269] (74) Thymidine 5′-(α-D-altropyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.0699 (M+H).

[0270] (75) Uridine 5′-(α-D-altropyranosyl diphosphate). HRMS (FAB):calc 565.0507; found m/z 565.0511 (M+H).

[0271] (76) Thymidine 5′-(α-D-gulopyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.00712 (M+H).

[0272] (77) Uridine 5′-(α-D-gulopyranosyl diphosphate). HRMS (FAB): calc565.0507; found m/z 565.0512 (M+H).

[0273] (78) Thymidine 5′-(α-D-idopyranosyl diphosphate). HRMS (FAB) calc563.0705; found m/z 563.0708 (M+H).

[0274] (79) Uridine 5′-(α-D-idopyranosyl diphosphate). HRMS (FAB): calc565.0507; found m/z 565.0507 (M+H).

[0275] (80) Thymidine 5′-(α-D-talopyranosyl diphosphate). HRMS (FAB)calc 563.0705; found m/z 563.0710 (M+H).

[0276] (81) Uridine 5′-(α-D-talopyranosyl diphosphate). HRMS (FAB): calc565.0507; found m/z 565.0499 (M+H).

[0277] Enzyme Purification.

[0278]E. coli-prfbA-C (from Professor Hung-wen Liu (Dept. of Chem.,Univ. of MN)) was grown in 2 L superbroth, 100 μg mL⁻¹ ampicillindivided among two 4 L baffled flasks for 18 hours at 37° C. Cells wereharvested by centrifugation (5000×g, 20 min, 4° C.), washed twice withbuffer A (50 mM potassium phosphate buffer, 1 mM EDTA, pH 7.5),resuspended in buffer A (4×weight) and split into two equal volumes.Each was sonicated by three 40 second bursts at 0° C. followed bycentrifugation (4400×g, 20 min, 4° C.) to remove cellular debris and afurther 1.3-fold dilution of the supernatant with buffer A. To thecombined supernatant (167 mL) was added 31.5 mL 5% streptomycin sulfatein a dropwise fashion followed by gentle stirring (1 hr, 4° C.) andcentrifugation (14,000×g, 30 min, 4° C.) to remove precipitate. Thesupernatant was diluted (0.1-fold 1M potassium phosphate buffer, pH 7.5)followed by the slow addition of ammonium sulfate crystals to 65%saturation, gentle stirring (7.5 hr, 4° C.) and centrifugation (4200×g,30 min, 4° C.). The precipitated protein was dissolved in a minimumamount of buffer A and dialyzed against buffer B (20 mM Tris.HCl, 1 mMEDTA, pH 7.5). The dialysate was applied to a column of DE52 (3 cm×15cm) which was washed with 50 mL buffer B and then eluted with a lineargradient (buffer B, 0-500 mM NaCl, 1.0 mL min⁻¹). The E_(p) fractions(which eluted in the range of 35-75 mM NaCl) were combined (24 mL) andconcentrated to 1 mL. Aliquots (300 μL) were further resolved by FPLC(S-200, 20×70 cm, 50 mM Tris.HCl, 200 mM NaCl, pH 7.5). The E_(p)fractions were combined (7 mL), concentrated (64 mg min⁻¹) and stored inaliquots (5, 20, and 200 μL) at −80° C. until their use.

[0279] General Methods.

[0280] Infrared spectra were recorded on a Perkin Elmer 1600 series FTIRspectrophotometer. ¹H NMR spectra were obtained on a Bruker AMX 400 (400MHz) and are reported in parts per million (δ) relative to eithertetramethylsilane (0.00 ppm) or CDCl₃ (7.25 ppm) for spectra run inCDCl₃ or relative to D₂O (4.82 ppm) or CD₃OD (3.35 ppm) for spectra runin D₂O. Coupling constants (J) are reported in hertz. ¹³C NMR arereported in δ relative to CDCl₃ (77.00 ppm) or CD₃OD (49.05 ppm) as aninternal reference and ³¹P NMR spectra are reported in δ relative toH₃PO₄ (0.00 ppm in D₂O). Routine mass spectra were recorded on a PESCIEX API 100 LC/MS mass spectrometer and HRMS was accomplished by theUniversity of California, Riverside Mass Spectrometry Facility. Opticalrotations were recorded on a Jasco DIP-370 polarimeter using a 1.0 dmcell at the room temperature (25° C.) and the reported concentrations.Melting points were measured with Electrothermal 1A-9100 digital meltingbpoint instrument. Chemicals used were reagent grade and used assupplied except where noted. Analytical TLC was performed on Whatman ALSil G/UV silica gel 60 plates. Compounds were visualized by sprayingI₂/KI/H₂SO₄ or by dipping the plates in a cerium sulfate-ammoniummolybdate solution followed by heating. Liquid column chromatography wasperformed using forced flow of the indicated solvent on E. Merck silicagel 60 (40-63 μm) and high pressure liquid chromatography was performedon a RAININ Dynamax SD-200 controlled with Dynamax HPLC software.

[0281] Although the above methods of IR, NMR, Mass Spec, and HRMS werethose used in these examples of the present invention, as indicatedabove, it would be known to those skilled in the art that otherinstruments, conditions, types of techniques, and the like may be usedfor visualization of compounds, to identify compounds and determinetheir concentrations and purity.

[0282] General Strategy for Azide Formation.

[0283] Azides in accordance with the present invention may be formedaccording to the following method. Protected glycoside is dissolved inCH₂Cl₂. The mixture is cooled to about 0° C. and pyridine and (CF₃SO₂)₂Oare added. The reaction was stirred for approximately 30 min at about 0°C. and then diluted with CH₂Cl₂. The organics were washed with water,dried over Na₂SO₄ and concentrated. The resulting crude residue wasdissolved, preferably in anhydrous DMF, to which was added NaN₃. Thereaction was subsequently stirred, preferably overnight, at roomtemperature and then diluted with EtOAc. The organics were washed withwater, dried over Na₂SO₄ and concentrated. Preferably, productpurification was accomplished by flash chromatography.

[0284] Other conditions, reagents, solutions and the like of the presentmethod, or other methods known to those skilled in the art may be usedin accordance with the present invention.

[0285] In a typical reaction, the appropriately protected glycoside (2.1mmol) was dissolved in 10 mL of CH₂Cl₂. The mixture was cooled to 0° C.to which was added pyridine (6.3 mmol) and (CF₃SO₂)₂O (3.2 mmol). Thereaction was stirred 30 min at 0° C. and then diluted with CH₂Cl₂ (150mL). The organics were washed with water (30 mL), dried over Na₂SO₄ andconcentrated. The resulting crude residue was dissolved in 10 mLanhydrous DMF, to which was added NaN₃ (407 mg, 6.3 mmol). The reactionwas subsequently stirred overnight at room temperature and then dilutedwith EtOAc (250 mL). The organics were washed with water (2×30 mL),dried over Na₂SO₄ and concentrated. Product purification wasaccomplished by flash chromatography (4:1 hexane/EtOAc).

[0286] Ethyl4-azide-2,3,6-tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside (FIG. 3(b)(94)).

[0287] Compound (AGCH) 93 (310 mg, 0.63 mmol)⁸ gave 285 mg (88%) desiredproduct. [α]_(D)=62.3° (c=1, CHCl₃); ¹H NMR (CDCl₃) 7.40-7.31 (m, 15H),4.94 (d, 1H, J=9.5 Hz), 4.92 (d, 1H, J=10.3 Hz), 4.84 (d, 1H, J=10.6Hz), 4.73 (d, 1H, J=10.3 Hz), 4.64 (d, 1H, J=12.0 Hz), 4.56 (d, 1H,J=12.0 Hz), 4.44 (d, 1H, J=9.6 Hz), 3.77 (dd, 1H, J=1.8, 10.9 Hz),3.71-3.62 (m, 2H), 3.54 (t, 1H, J=9.4 Hz), 3.45 (t, 1H, J=9.8 Hz), 3.3(m, 1H), 2.84-2.69 (m, 2H), 1.33 (t, 3H, J=7.4 Hz); ¹³C NMR (CDCl₃)137.8, 137.6, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.5, 85.0,84.6, 81.3, 77.8, 75.5, 75.3, 73.3, 69.1, 61.9, 24.8, 15.0; MS: calcdfor C₂₉H₃₃N₃O₄SNa 542.2, found m/z 542.0 (M+Na).

[0288] Ethyl4-azide-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-1-thio-β-D-glucopyranoside(FIG. 3(b) (106)).

[0289] Compound (FIG. 3(b) (105)) gave 0.78 g (87.4%) substantially pureproduct. [α]_(D)=38° (c=1, CHC₃); ¹H NMR (CDCl₃) 8.03 (d, 2H, J=8.2 Hz),7.60 (m, 1H), 7.47 (t, 2H, J=7.5 Hz), 7.30 (s, 2H), 7.12 (m, 3H), 5.43(t, 1H, J=9.8 Hz), 4.80 (d, 1H, J=10.7 Hz), 4.34 (m, 2H), 3.54 (t, 1H,J=9.5 Hz), 3.44 (m, 1H), 3.32 (t, 1H, J=9.9 Hz), 2.79 (m, 2H), 1.42 (d,3H, J=6.0 Hz), 1.34 (t, 3H, J=7.4 Hz); ¹³C NMR (CDCl₃) 165.9, 137.5,133.8, 130.2, 129.8, 128.9, 128.7, 128.6, 128.2, 85.4, 79.6, 77.7, 76.7,75.4, 75.1, 66.7, 25.7, 19.0, 15.4; MS: calcd for C₂₂H₂₅N₃O₄SNa 450.1,found m/z 450.0 (M+Na).

[0290] Ethyl 3-O-benzoyl-2-O-benzyl-6-deoxy-1-thio-β-D-galactopyranoside(FIG. 3(b) (105)).

[0291] Ethyl 2,3,4-tri-O-acetyl-6-deoxy-1-thio-β-D-galactopyranoside(FIG. 3(b) (104)), 2.72 g, 8.14 mmol) was dissolved in 30 mL MeOH towhich 1.2 mL 25% sodium methoxide was added. From this reaction, 1.58 g(93.3%) ethyl 6-deoxy-1-thio-β-D-glactopyranoside was obtained afterpurification which was combined with TsOH (140 mg, 0.73 mmol) and2,2-dimethoxypropane (1.9 mL, 15.4 mmol) in 15 mL anhydrous DMF. Thereaction was stirred overnight at room temperature, diluted with 200 mLEtOAc and washed successively with saturated NaHCO₃ solution (50 mL) andwater (30 mL). The organics were dried over Na₂SO₄ and purified viasilica gel chromatography (3:1 hexane/EtOAc) to afford 1.73 g (86%) ofpurified ethyl6-deoxy-3,4-O-isopropylidene-1-thio-α-D-galactopyranoside. [α]_(D)=11.9°(c=1, CHCl₃); ¹H NMR (CDCl₃) 4.19 (d, 1H, J=10.2 Hz), 4.01 (m, 2H), 3.84(dq, 1H, J=1.7, 13.1 Hz), 3.50 (dd, 1H, J=6.2, 10.2 Hz), 2.71 (m, 2H),1.59 (s, 3H), 1.37 (d, 3H, J=6.6 Hz), 1.33 (s, 3H), 1.28 (t, 3H, J=7.5Hz); ¹³C NMR (CDCl₃) 110.2, 85.5, 79.5, 76.8. 73.2, 72.3, 28.6, 26.7,24.6, 17.2, 15.6; MS: calcd for C₁₁H₂₀O₄SNa 271.1, found m/z 270.9(M+Na).

[0292] The obtained ethyl6-deoxy-3,4-O-isopropylidene-1-thio-α-D-galactopyranoside (1.50 g, 6.0mmol) was combined with of 60% sodium hydride (0.36 g, 9 mmol) andbenzyl bromide (1.44 mL, 12.1 mmol) in 20 mL dry DMF. The reaction wasstirred overnight and 1.7 g (83%) ethyl2-O-benzyl-6-deoxy-3,4-O-isopropylidene-1-thio-α-D-galactopyranoside wasobtained after the typical work up and purification. [α]_(D)=−2.8° (c=1,CHCl₃); ¹H NMR (CDCl₃) 7.35 (d, 2H, J=7.1 Hz), 7.25 (t, 2H, J=7.1 Hz),7.09 (m, 1H), 4.77 (d, 1H, J=11.4 Hz), 4.69 (d, 1H, J=11.4 Hz), 4.31 (d,1H, J=9.8 Hz), 4.11 (m, 1H), 3.96 (dd, 1H, J=2.0, 15.6 Hz), 3.73 (m,1H), 3.36 (dd, 1H, J=6.7, 19.8 Hz), 2.64 (m, 2H), 1.42 (s, 3H), 1.29 (d,3H, J=6.6 Hz), 1.27 (s, 3H), 1.22 (t, 3H, J=7.4 Hz); ¹³C NMR (CDCl₃)137.9, 128.3, 128.2, 127.6, 109.5, 83.3, 79.7, 78.0, 76.5, 73.4, 72.4,28.0, 26.4, 24.4, 16.8, 14.8; MS: calcd for C₁₈H₂₆O₄SNa 361.1, found m/z361.0 (M+Na).

[0293] The obtained ethyl2-O-benzyl-6-deoxy-3,4-O-isopropylidene-1-thio-β-D-galactopyranoside(1.82 g, 5.38 mmol) was dissolved in a mixture solution including 15 mL0.5M HCl and 45 mL MeOH and the mixture was subsequently refluxed for 30min. The reaction was cooled to room temperature, neutralized with solidNaHCO₃, and the resulting mixture concentrated. The concentrate wasdiluted with EtOAc (250 mL), washed with water (2×20 mL) and brine (20mL), dried over Na₂SO₄ and purified by flash chromatography (1:1hexane/EtOAc) to give 1.51 g (94%) substantially pure ethyl2-O-benzyl-6-deoxy-1-thio-β-D-galactopyranoside. [α]_(D)=8.4° (c=1,CHCl₃); ¹H NMR (CDCl₃) 7.42-7.29 (m, 5H), 4.97 (d, 1H, J=11.0 Hz), 4.67(d, 1H, J=11.0 Hz), 4.40 (d, 1H, J=9.6 Hz), 3.75 (m, 1H), 3.61 (m, 2H),3.45 (t, 1H, J=9.3 Hz), 2.78 (m, 2H), 2.48 (d, 1H, J=5.0 Hz), 2.14 (d,1H, J=5.0 Hz), 1.32 (m, 6H); ¹³C NMR (CDCl₃) 138.5, 129.0, 128.7, 128.5,85.1, 79.3, 77.6, 75.7, 75.6, 74.8,72.2, 25.4, 16.9, 15.4; MS: calcd forC₁₅H₂₂O₄SNa 321.1, found m/z 321.0 (M+Na).

[0294] To a solution of ethyl2-O-benzyl-6-deoxy-1-thio-β-D-galactopyranoside (1.03 g, 3.45 mmol) andDMAP (126 mg, 1.0 mmol) in 10 mL of dry CH₂Cl₂ at −30° C. was added Et₃N(1.92 mL, 13.8 mmol). Benzoyl chloride (0.4 mL, 3.45 mmol) was added tothis mixture in a dropwise fashion, and the stirred at −30° C. for 3 hr.The reaction was then quenched by the addition of MeOH (2 mL) and themixture was gradually warmed to room temperature after which theresulting mixture was diluted with EtOAc (250 mL). The solution waswashed with saturated NaHCO₃ solution (2×20 mL), water (30 mL), driedover Na₂SO₄, concentrated and purified by flash chromatography (3:1 to1:1 hexane/EtOAc) to give 1.12 g (80%) of the title product.[α]_(D)=96.9° (c=1, CHCl₃); ¹H NMR (CDCl₃) 8.09-8.03 (m, 2H), 7.56 (t,1H, J=7.4 Hz), 7.49 (m, 2H), 7.22 (m, 2H), 7.18 (m, 3H), 5.28 (dd, 1H,J=3.0, 9.6 Hz), 4.87 (d, 1H, J=10.6 Hz), 4.67 (d, 1H, J=10.6 Hz), 4.56(d, 1H, J=9.7 Hz), 4.12 (m, 1H), 3.85 (t, 1H, J=9.8 Hz), 3.80 (m, 1H),2.81 (m, 2H), 1.93 (d, 1H, J=6.7 Hz), 1.36 (m, 6H); ¹³C NMR (CDCl₃)166.2, 138.0, 133.7, 130.2, 130.1, 128.9, 128.7, 128.6, 128.2, 85.7,78.1, 77.6, 76.5, 76.0, 74.7, 70.9, 25.6, 16.9, 15.4; MS: calcd forC₂₂H₂₆O₅SNa 425.1, found m/z 425.2 (M+Na).

[0295] Strategy for Formation of Protected Ethyl1-thio-β-D-hexopyranosides.

[0296] Ethyl 1-thio-β-D-hexopyranosides may be generally formed as setforth above. The following method is another exemplary embodiment ofsuch method used in accordance with the present invention. In a typicalreaction, a mixture of 4.0 mmol protected monosaccharide, 1.5 mL(ethylthio)trimethylsilane (8.0 mmol) and 1.95 g zinc iodide (7.8 mmol)in 30 mL dry dichloromethane was refluxed for 30 min under argonatmosphere. The reaction was then cooled, 50 mL water was added afterwhich the mixture was extracted with chloroform (3×50 mL). The combinedorganic extracts were washed successively with water (30 mL), saturatedNaHCO₃ solution (30 mL) and brine (30 mL). The organics were dried overNa₂SO₄, concentrated and resolved by silica gel chromatography (2:1hexanes/EtOAc) to give the desired product.

[0297] Again, variations of this method may be made in accordance withthe present invention, as would be apparent to one skilled in the art.

[0298] Ethyl2,4,6-tri-O-acetyl-3-azide-3-deoxy-1-thio-β-D-glucopyranoside (FIG. 3(b)(99)).

[0299] Compound (FIG. 3(b) (99)) (1.5 g, 4.0 mmol) gave 1.26 g (83.5%)title compound. [α]_(D)=−49.4° (c=0.5, CHCl₃); ¹H NMR (CDCl₃) 4.95 (m,2H), 4.43 (d, 1H, J=9.9 Hz), 4.19 (dd, 1H, J=4.1, 12.4 Hz), 4.09 (m,1H), 3.65 (m, 2H), 2.68 (m, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 1.24 (t,3H, J=7.5 Hz), 2.06 (s, 3H); ¹³C NMR (CDCl₃) 171.0, 169.6, 169.6, 84.2,76.8, 70.3, 68.7, 66.1, 62.6, 24.4, 21.2, 21.1, 21.0, 15.1; MS: calcdfor C₁₄H₂₁N₃O₇SNa 398.1, found m/z 397.9 (M+Na).

[0300] Ethyl2,3,4-tri-O-acetyl-6-azide-6-deoxy-1-thio-β-D-glucopyranoside (FIG. 3(b)(88)).

[0301] Compound (FIG. 3(b) (87)) (680 mg, 1.8 mmol)⁶ gave 590 mg (86%)of the desired title compound. [α]_(D)=−17.5° (c=1, CHCl₃); ¹H NMR(CDCl₃) 5.23 (t, 1H, J=9.4 Hz), 5.02 (m, 2H), 4.54 (d, 1H, J=10.0 Hz),3.62 (m, 1H), 3.37 (dd, 1H, J=6.5, 13.5 Hz), 3.30 (dd, 1H, J=2.8, 13.5Hz), 2.73 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.28 (t,3H, J=7.4 Hz); ¹³C NMR (CDCl₃) 170.0, 169.4, 169.2, 83.0, 77.2, 73.6,69.7, 69.3, 51.0, 23.6, 20.6, 20.5, 14.6. MS: calcd for C₁₄H₂₁N₃O₇SNa398.1, found m/z 397.5 (M+Na).

[0302] Ethyl 2,3,4-tri-O-acetyl-6-deoxy-1-thio-β-D-galactopyranoside(FIG. 3(b) (104)).

[0303] Compound (FIG. 3(b) (103)) (6.1 mmol) gave 1.73 g (83%) of thesubstantially pure product. [α]_(D)=−17.5° (c=1, CHCl₃); ¹H NMR (CDCl₃)5.28 (d, 1H, J=3.3 Hz), 5.22 (t, 1H, J=9.9 Hz), 5.05 (dd, 1H, J=3.4, 9.9Hz), 4.46 (d, 1H, J=9.9 Hz), 3.82 (dd, 1H, J=6.4, 12.8 Hz), 2.74 (m,2H), 2.17 (s, 3H), 2.06 (s, 3H), 1.98 (s, 3H), 1.28 (t, 3H, J=7.4 Hz),1.22 (d, 3H, J=6.4 Hz); ¹³C NMR (CDCl₃) 171.0, 170.6, 170.1, 83.9, 77.6,73.6, 72.7, 70.8, 67.7, 24.5, 21.3, 21.1, 21.0, 16.8, 15.1; MS: calcdfor C₁₄H₂₂O₇SNa 357.1, found m/z 356.6 (M+Na).

[0304] General Strategy for O-Acetyl to O-Benzyl Conversion.

[0305] O-Acetyl may be converted to O-Benzyl according to the followingmethod or other methods known to those skilled in the art. Protectedethyl 1-thio-β-D-hexopyranoside was dissolved in dry MeOH and toluene towhich a sodium methoxide solution is added. The mixture was stirred forabout 2½ M to about 3½ hrs. at room temperature and neutralized. Theorganics are then concentrated and the corresponding crude unprotected1-ethylthio-β-D-glucopyranoside directly dissolved in dry DMF. To thismixture NaH and benzyl bromide is added. The reaction is stirred at roomtemperature, preferably overnight. The mixture was then diluted withEtOAc, washed with H₂O, brine and organics, dried, concentrated, andpurified to give the purified product.

[0306] Other conditions, reagents, solutions and the like of the presentmethod, or other methods known to those skilled in the art may be usedin accordance with the present invention.

[0307] In a typical reaction, 2.8 mmol of protected ethyl1-thio-β-D-hexopyranoside was dissolved in 20 mL dry MeOH and 5 mLtoluene to which 0.5 mL of a sodium methoxide solution (25% NaOMe inmethanol) was added. The mixture was stirred for 3 hr at roomtemperature and neutralized with DOWEX 50W X8-100 resin. The organicswere concentrated and the corresponding crude unprotected1-ethylthio-β-D-glucopyranoside directly dissolved in 15 mL dry DMF. Tothis mixture 330 mg 60% NaH (8.25 mmol) and 1.6 mL benzyl bromide wasadded. The reaction was stirred at room temperature overnight. Themixture was then diluted with 200 mL EtOAc, washed with H₂O (2×30 mL),brine (30 mL) and the organics dried over Na₂SO₄, concentrated andpurified by silica gel chromatography (8:1 hexane/EtOAc) to give thepurified product.

[0308] Ethyl3-azide-2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (FIG. 3(b)(100)).

[0309] Compound (FIG. 3(b) (99)) (1.05 g, 2.8 mmol) gave 1.03 g (71%) ofthe desired title compound. [α]_(D)=−13.6° (c=1, CHCl₃); ¹H NMR (CDCl₃)7.36-7.28 (m, 15H), 4.90 (d, 1H, J=10.1 Hz), 4.79 (d, 1H, J=10.6 Hz),4.74 (d, 1H, J=10.1 Hz), 4.60 (d, 1H, J=12.1 Hz), 4.54-4.47 (m, 2H),4.43 (d, 1H, J=9.6 Hz), 3.70 (m, 1H), 3.57 (t, 1H, J=9.1 Hz), 3.45 (m,2H), 3.26 (t, 1H, J=9.5 Hz), 2.75 (m, 2H), 1.32 (t, 1H, J=7.3 Hz); ¹³CNMR (CDCl₃) 138.4, 137.9, 137.8, 129.5, 129.2, 129.1, 128.9, 128.8,128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.7, 85.7,80.4, 79.7, 76.7, 75.8, 75.3, 73.9, 72.5, 71.0, 69.1, 25.6, 15.6; MS:calcd for C₂₉H₃₃N₃O₄SNa 542.2, found m/z 542.0 (M+Na).

[0310] Ethyl6-azide-2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (FIG. 3(b)(89)).

[0311] Compound (FIG. 3(b) (88)) (560 mg, 1.5 mmol) gave 645 mg (85%) ofthe desired product. [α]_(D)=7.9° (c=1, CHCl₃); ¹H NMR (CDCl₃) 7.32-7.16(m, 15H), 4.87 (d, 1H, J=10.9 Hz), 4.86 (d, 1H, J=10.2 Hz), 4.79 (d, 1H,J=11.2 Hz), 4.76 (d, 1H, J=11.0 Hz), 4.66 (d, 1H, J=10.2 Hz), 4.50 (d,1H, J=11.0 Hz), 4.43 (d, 1H, J=9.8 Hz), 3.62 (m, 1H), 3.43-3.35 (m, 4H),3.24 (dd, 1H, J=6.0, 13.1 Hz), 2.72 (m, 2H), 1.26 (t, 3H, J=1.5 Hz); ¹³CNMR (CDCl₃) 138.2, 137.7, 137.5, 128.5, 128.4, 128.3, 128.2, 128.0,127.8, 127.7, 86.3, 84.6, 81.5, 78.4, 78.2, 75.7, 75.4, 75.1, 51.3,24.4, 14.9; MS: calcd for C₂₉H₃₃N₃O₄SNa 542.2, found m/z 541.9 (M+Na).

[0312] General Strategy for Conversion of Azides to Acetamides.

[0313] Azides may be converted to Acetamides according to the followingmethod or other methods known to those skilled in the art.Benzyl-protected ethyl 1-thio-β-D-azidodeoxy-hexopyranoside and SnCl₂are combined in acetonitrile. To this mixture thiophenol and Et₃N areadded and the reaction is stirred for about ½ to about 1½ hr at roomtemperature. The mixture is then diluted with EtOAc and washed,preferably with 2N NaOH, water, and brine. The organics are dried,preferably over Na₂SO₄, concentrated to dryness and the crude residuedissolved in dry pyridine. To this mixture 2 mL acetic anhydride isadded and the reaction stirred, preferably overnight, at roomtemperature. The reaction is concentrated and purified directly bysilica gel chromatography (3:2 to 1:1 hexane/EtOAc) to give the purifiedproduct.

[0314] Other conditions, reagents, solutions and the like of the presentmethod, or other methods known to those skilled in the art may be usedin accordance with the present invention.

[0315] In a typical reaction, benzyl-protected ethyl1-thio-β-D-azidodeoxyhexopyranoside (2.8 mmol) and SnCl₂ (1.73 mmol)were combined in 10 mL of acetonitrile. To this mixture thiophenol (6.9mmol) and Et₃N (5.2 mmol) were added and the reaction was stirred for 1hr at room temperature under argon atmosphere. The mixture was thendiluted with EtOAc (150 mL) and washed with 2N NaOH (2×2 mL), water (20mL) and brine (30 mL). The organics were dried over Na₂SO₄, concentratedto dryness and the crude residue dissolved in 10 mL dry pyridine. Tothis mixture 2 mL acetic anhydride was added and the reaction stirredovernight at room temperature. The reaction was concentrated andpurified directly by silica gel chromatography (3:2 to 1:1 hexane/EtOAc)to give the purified product.

[0316] Ethyl3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (FIG.3(b) (101)).

[0317] Compound (FIG. 3(b) (100)) (600 mg, 1.15 mmol) gave 523 mg (85%)of the desired product. [α]_(D)=−5.4° (c=1, CHCl₃); ¹H NMR (CDCl₃)7.27-7.17 (m, 15H), 5.57 (d, 1H, J=8.6 Hz), 4.73 (d, 1H, J=11.2 Hz),4.55-4.39 (m, 5H), 3.97 (dd, 1H, J=8.3, 16.5 Hz), 3.66 (m, 2H), 3.58(dd, 1H, J=4.1, 10.8 Hz), 3.50 (m, 1H), 3.44 (t, 1H, J=8.4 Hz), 2.71 (m,2H), 1.60 (s, 3H), 1.24 (t, 3H, J=7.4 Hz); ¹³C NMR (CDCl₃) 170.0, 137.9,137.8, 137.7, 128.7, 128.3, 128.2, 128.0, 127.9, 127.7, 127.6, 85.3,80.1, 78.8, 75.4, 73.7, 73.5, 73.1, 69.4, 55.7, 25.2, 23.4, 15.0; MS:calcd for C₃₁H₃₇NO₅SNa 558.2, found m/z 558.0 (M+Na).

[0318] Ethyl4-acetamido-2,3,6-tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside (FIG.3(b) (95)).

[0319] Compound (FIG. 3(b) (94)) (640 mg, 1.23 mmol) gave 530 mg desiredproduct (80%) [α]_(D)=−36.6° (c=0.5, CHCl₃); ¹H NMR (CDCl₃) 7.32-7.10(m, 15H), 5.13 (br, 1H), 4.85 (d, 1H, J=10.2 Hz), 4.75 (d, 1H, J=11.7Hz), 4.70 (d, 1H, J=10.7 Hz), 4.64 (d, 1H, J=10.2 Hz), 4.43 (m, 3H),3.64-3.47 (m, 5H), 3.37 (m, 1H), 2.67 (m, 2H), 1.61 (s, 3H), 1.25 (t,3H, J=7.4 Hz); ¹³C NMR (CDCl₃) 170.3, 138.3, 138.0, 137.8, 128.5, 128.3,128.2, 128.1, 127.8, 127.7, 127.6, 84.9, 82.1, 81.7, 78.1, 75.3, 74.7,73.4, 70.0, 52.6, 24.9, 23.3, 15.1; MS: calcd for C₃₁H₃₇NO₅SNa 558.2,found m/z 557.9 (M+Na).

[0320] Ethyl6-acetamido-2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (FIG.3(b) (90)).

[0321] Compound (FIG. 3(b) (89)) (502 mg, 0.97 mmol) gave 450 mg desiredproduct (87%). [α]_(D)=−20.4° (c=1.0, CHCl₃); ¹H NMR (CDCl₃) 7.51-7.23(m, 15H), 5.87(d, 1H, J=4.6 Hz), 4.99-4.78 (m, 4H), 4.73 (d, 1H, J=10.2Hz), 4.63 (d, 1H, J=10.4 Hz), 4.45 (d, 1H, J=9.8 Hz), 3.70-3.60 (m, 2H),3.52 (m, 1H), 3.41-3.34 (m, 3H), 2.74 (m, 2H), 1.95 (s, 3H), 1.32 (t,3H, J=7.4 Hz); ¹³C NMR (CDCl₃) 169.8, 138.1, 137.6, 137.5, 128.4, 128.3,128.2, 128.1, 127.9, 127.8, 127.6, 86.2, 85.1, 81.5, 78.5, 77.1, 75.6,75.4, 75.1, 39.9, 25.2, 23.1, 15.1; MS: calcd for C₃₁H₃₇NO₅SNa 558.2,found m/z 558.2 (M+Na).

[0322] Phosphorylation Procedure.

[0323] As set forth in the methods above, phosphorylation according tothe present invention may occur via a protected ethyl1-thio-β-D-hexopyranoside. The following method is another exemplaryembodiment of such method used in accordance with the present invention.

[0324] In a typical reaction, 1.13 mmol protected ethyl1-thio-β-D-hexopyranoside and 1.7 mmol dibenzyl phosphate wereco-evaporated two times from dry toluene and further dried under highvacuum for 4 hr to which 1.36 mmol of N-iodosuccinamide and 500 mg ofdry molecular sieves were added. The mixture was then dissolved in 10 mLdry dichloromethane, cooled to −30° C. and 30 μL oftrifluoromethanesulfonic acid (0.34 mmol) was added. The reaction wasmaintained at −30° C. for 30 min with stirring and then diluted withEtOAc (150 mL), washed with saturated Na₂S₂O₃ (20 mL), saturated NaHCO₃(20 mL), H₂O (20 mL), and brine (30 mL). The organics were dried overNa₂SO₄, filtered, concentrated and purified by chromatography on silicagel (3:1 hexane/EtOAc) to give the desired product.

[0325] Again, variations of this method may be made in accordance withthe present invention, as would be apparent to one skilled in the art.Dibenzyl-(3-azide-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)phosphate (FIG. 3(b) (100a)).

[0326] Compound (FIG. 3(b) (100)) (590 mg, 1.55 mmol) gave 700 mg (84%)of the title compound. [α]_(D)=57.8° (c=1, CHCl₃); ¹H NMR (CDCl₃)7.45-7.27 (m, 25H), 5.99 (dd, 1H, J=3.2, 6.8 Hz), 5.11-5.05 (m, 4H),4.84 (d, 1H, J=10.6 Hz), 4.82 (d, 1H, J=11.4 Hz), 4.72 (d, 1H, J=11.5Hz), 4.60 (d, 1H, J=12.0 Hz), 4.49 (d, 1H, J=10.7 Hz), 4.46 (d, 1H,J=12.1 Hz), 3.84 (m, 2H), 3.68 (dd, 1H, J=3.0, 10.9 Hz), 3.57 (t, 1H,J=9.8 Hz), 3.48 (m, 2H); ¹³C NMR (CDCl₃) 137.9, 137.6, 137.4, 136.2,136.1, 136.0, 129.0, 128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1,128.0, 94.9, 77.4, 76.1, 75.7, 75.3, 74.0, 73.2, 72.4, 69.9, 69.8, 69.7,69.6, 67.9, 65.2; ³¹P NMR (CDCl₃) 0.82; MS: calcd for C₄₁H₄₂N₃O₈PNa758.2, found m/z 758.2 (M+Na).

[0327]Dibenzyl-(3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)Phosphate (FIG. 3(b) (101a)).

[0328] Compound (FIG. 3(b) (101)) (490 mg, 0.91 mmol) gave 480 mg (70%)of the desired product. [α]_(D)=52° (c=1, CHCl₃); ¹H NMR (CDCl₃)7.40-7.19 (m, 25H), 5.94 (dd, 1H, J=3.2, 6.7 Hz), 5.07 (br, 1H), 4.97(m, 4H), 4.63 (d, 1H, J=11.7 Hz), 4.56 (d, 1H, J=12.0 Hz), 4.39 (m, 4H),3.90 (m, 2H), 3.85 (m, 2H), 3.56 (dd, 1H, J=3.3, 11.0 Hz), 3.39 (dd, 1H,J=1.6, 11.0 Hz), 1.76 (s, 3H); ¹³C NMR (CDCl₃) 170.7, 137.8, 137.5,137.4, 135.6, 135.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6,127.5, 94.9, 77.2, 75.1, 74.0, 73.3, 72.8, 72.3, 69.3, 69.4, 69.0, 67.9,53.4, 23.4; ³¹P NMR (CDCl₃) 0.62; MS: calcd for C₄₃H₄₆NO₉PNa 774.3,found m/z 774.3 (M+Na).

[0329] Dibenzyl-(4-azide-2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl)Phosphate. (FIG. 3(b) (94a))

[0330] Compound (FIG. 3(b) (94)) (280 mg, 054 mmol) gave 316 mg (80%) ofthe desired product. [α]_(D)=105.8° (c=1, CHCl₃); 7.28-7.14 (m, 25H),5.85 (dd, 1H, J=3.2, 6.8 Hz), 5.12-4.96 (m, 5H), 4.82 (d, 1H, J=10.6Hz), 4.71-4.66 (m, 2H), 4.57 (d, 1H, J=11.3 Hz), 4.50 (d, 1H, J=12.1Hz), 4.37 (d, 1H, J=12.1 Hz), 3.68-3.47 (m, 5H), 3.37 (dd, 1H, J=1.5,11.0 Hz); ¹³C NMR (CDCl₃) 137.7, 137.6, 137.5, 135.7, 135.6, 128.5,128.4, 128.3, 128.2, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5,95.4, 78.9, 78.8, 75.6, 75.5, 73.4, 72.9, 71.5, 69.3, 69.2, 69.2, 67.9,60.8; ³¹P NMR (CDCl₃) 0.82; MS: calcd for C₄₁H₄₂N₃O₈PNa 758.2, found m/z758.0 (M+Na).

[0331]Dibenzyl-(4-acetamido-2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl)Phosphate. (FIG. 3(b) (95a))

[0332] Compound (FIG. 3(b) (95)) (430 mg, 0.80 mmol) gave 389 mg (65%)of the desired product. [α]_(D)=35° (c=1, CHCl₃); ¹H NMR (CDCl₃)7.23-7.09 (m, 25H), 5.82 (dd, 1H, J=3.2, 6.8 Hz), 5.47 (d, 1H, J=8.5Hz), 5.01-4.93 (m, 4H), 4.69 (d, 1H, J=11.7 Hz), 4.59 (d, 1H, J=11.1Hz), 4.54 (m, 2H), 4.35 (d, 1H, J=11.9 Hz), 4.30 (d, 1H, J=11.9 Hz),3.92 (m, 1H), 3.86 (m, 1H), 3.77 (t, 1H, J=9.7 Hz), 3.55 (m, 1H), 3.39(m, 2H), 1.65 (s, 3H); ¹³C NMR (CDCl₃) 170.0, 138.2, 137.7, 137.3 135.7,135.6, 135.5 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,127.4, 95.6, 79.4, 79.3, 77.2, 74.5. 73.3, 72.8, 72.2, 69.2, 50.8, 23.2;³¹P NMR (CDCl₃) 0.71; MS: calcd for C₄₃H₄₆NO₉PNa 774.3, found m/z 774.3(M+Na)

[0333] Dibenzyl-(6-azide-2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl)Phosphate. (FIG. 3(b) (89a))

[0334] Compound (FIG. 3(b) (89)) (430 mg, 0.76 mmol) gave 285 mg (51%)of the desired product and 160 mg the β isomer. [α]_(D)=41.5° (c=1,CHCl₃); ¹H NMR(CDCl₃) 7.28-7.16 (m, 25H), 5.87 (dd, 1H, J=3.2, 6.7 Hz),5.05-4.92 (m, 4H), 4.85 (d, 1H, J=10.9 Hz), 4.81 (d, 1H, J=11.0 Hz),4.71 (d, 1H, J=11.3 Hz), 4.70 (d, 1H, J=10.9 Hz), 4.60 (d, 1H, J=11.3Hz), 4.50 (d, 1H, J=11.0 Hz), 3.81 (m, 2H), 3.54 (dt, 1H, J=9.5, 3.1Hz), 3.47 (t, 1H, J=9.5 Hz), 3.18 (m, 2H); ¹³C NMR (CDCl₃) 138.2, 137.7,137.3, 135.7, 135.6, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7,127.6, 95.1, 80.7, 79.3, 79.2, 77.1, 75.5, 75.1, 73.0, 71.9, 69.3, 69.2,69.2, 69.1, 50.7; ³¹P NMR (CDCl₃) 0.75; MS: calcd for C₄₁H₄₂N₃O₈PNa758.2, found m/z 758.1 (M+Na).

[0335]Dibenzyl-(6-acetamido-2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl)Phosphate. (FIG. 3(b) (90a))

[0336] Compound (FIG. 3(b) (90)) (430 mg, 0.80 mmol) gave 389 mg (65.0%)of the desired product. ¹H NMR (CDCl₃) 7.27-7.19 (m, 25H), 6.00 (br,1H), 5.68 (dd, 1H, J=3.4, 5.5 Hz), 4.99-4.93 (m, 4H), 4.85(d, 1H, J=11.9Hz), 4.76 (d, 1H, J=10.6 Hz), 4.72 (d, 1H, J=10.5 Hz), 4.65 (d, 1H,J=11.5 Hz), 4.60 (d, 1H, J=11.5 Hz), 4.57 (d, 1H, J=10.5 Hz), 3.81 (m,2H), 3.49 (dt, 1H, J=3.5, 9.4 Hz), 3.44 (m, 2H), 3.24 (t, 1H, J=9.5 Hz);¹³C NMR (CDCl₃) 178.5, 138.3, 138.0, 137.9, 136.2, 136.1, 129.0, 128.9,128.8, 128.5, 128.4, 128.3, 128.1, 95.5, 75.7, 75.6, 74.6, 73.9, 73.4,72.9, 70.0, 69.9, 69.6, 69.5, 68.5, 54.0, 29.9; ³¹P NMR (CDCl₃) 0.53;MS: calcd for C₄₃H₄₆NO₉PNa 774.6, found m/z 774.3 (M+Na).

[0337]Dibenzyl-(4-azide-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-glucopyranosyl)Phosphate. (FIG. 3(b) (106a))

[0338] Compound (FIG. 3(b) (106)) (323 mg, 0.76 mmol) gave 350 mg (72%)substantially pure product. [α]_(D) =100.1° (c=1, CHCl₃); ¹H NMR (CDCl₃)8.16 (m, 2H), 7.62 (m, 1H), 7.49 (t, 2H, J=7.9 Hz), 7.34-714 (m, 11H),5.98 (dd, 1H, J=3.2, 7.1 Hz), 5.68 (t, 1H, J=9.8 Hz), 5.13-5.05 (m, 4H),4.68 (d, 1H, J=12.1 Hz), 4.50 (d, 1H, J=12.1 Hz), 3.81(m, 1H), 3.68 (dt,1H, J=3.0, 9.8 Hz), 3.28 (t, 1H, J=10.0 Hz), 1.24 (d, 3H, J=6.2 Hz); ¹³CNMR (CDCl₃) 185.8, 137.2, 136.1, 136.0, 133.8, 130.3, 129.9, 129.0,128.9, 128.8, 128.6, 128.4, 128.1, 95.1, 77.7, 76.5, 72.7, 72.1, 70.1,70.1, 69.7, 69.7, 68.7, 66.3, 18.6; ³¹P NMR (CDCl₃) 0.52; MS: calcd forC₃₄H₃₄N₃O₈PNa 666.2 found m/z 666.2 (M+Na).

[0339] Strategy for Final Deprotection and Conversion to the SodiumSalt.

[0340] Set forth above is a general strategy for final deprotection andconversion to the sodium salt according to the present invention. Thefollowing method is another exemplary embodiment of such method used inaccordance with the present invention.

[0341] In a typical reaction, the protected α-D-pyranosyl phosphate (0.5mmol) was dissolved in 15 mL MeOH, 1.5 mL 1N NaHCO₃ solution and 150 mg10% Pd/C were added. The mixture was stirred overnight at roomtemperature under hydrogen atmosphere after which the catalyst wasremoved by filtration and the filtrate concentrated and redissolved 10mL water. The aqueous layer was extracted with EtOAc (10 mL), and thensubmitted to an anion exchange column (Dowex 1×8, 1.2×12 cm) eluted with100 mL water, 100 mL 0.1 M NH₄HCO₃, 100 mL 0.2 M NH₄HCO₃ and 100 mL 0.3M NH₄HCO₃. The product eluted with 0.2M NH₄HCO₃ and these fractions werepooled and co-evaporated with ethanol several times to remove excessNH₄HCO₃. The obtained sugar phosphate ammonium salt was subsequentlydissolved in 5 mL water and applied to an AG-X8 cation-exchange column(Na⁺ type) eluted with 100 mL water. The product containing fractionswere collected and lyophilized to give the desired product as the sodiumsalt.

[0342] Again, variations of this method may be made in accordance withthe present invention, as would be apparent to one skilled in the art.

[0343] Disodium (3-amino-3-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (96)).

[0344] Dibenzyl-(3-azide-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)phosphate (250 mg, 0.34 mmol) gave 68 mg (66%) of the title compound.[α]_(D)=68.1° (c=1, H₂O); ¹H NMR (D₂O) 5.46 (dd, 1H, J=3.0, 7.0 Hz),3.93 (m, 1H), 3.85 (m, 1H), 3.74 (dd, 1H, J=4.5, 12.5 Hz), 3.69 (m, 1H),3.58 (m, 1H), 3.45 (t, 1H, J=10.2 Hz); ¹³C NMR (D₂O) 91.9, 71.2, 68.4,65.3, 59.3, 54.8; ³¹P NMR (D₂O) 2.85; HRMS: calcd for C₆H₁₃NO₈P258.0379, found m/z 258.0372 (M+H).

[0345] Disodium-(3-acetamido-3-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (97)).

[0346]Dibenzyl-(3-acetamido-2,4,6-tri-O-benzyl-3-deoxy-α-D-glucopyranosyl)phosphate (280 mg, 0.37 mmol) gave 59 mg (53%) of the desired product.[α]_(D)=93.6° (c=1, H₂O); ¹HNMR (D₂O) 5.43 (dd, 1H, J=2.9, 6.5 Hz), 4.07(t, 1H, J=10.3 Hz), 3.88 (dd, 1H, J=2.5, 9.7 Hz), 3.80 (m, 1H), 3.71(dd,1H, J=4.7, 12.3 Hz), 3.57 (m, 1H), 3.40(t, 1H, 10.1 Hz), 2.01(s, 3H);¹³C NMR (D₂O) 174.4, 92.8, 71.6, 69.5, 67.1, 59.8, 53.3, 21.5; ³¹P NMR(D₂O) 2.07; HRMS: calcd for C₈H₁₅NO₉P 300.0484, found m/z 300.0478(M+H).

[0347] Disodium-(4-amino-4-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (91)).

[0348] Dibenzyl-(4-azide-2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl)phosphate (350 mg, 0.476 mmol) gave 77 mg (54%) the desired product. ¹HNMR (D₂O) 5.46 (dd, 1H, J=3.2, 7.1 Hz), 4.11 (m, 1H), 3.90-3.75 (m, 3H),3.59 (m, 1H), 3.13 (t, 1H, J=10.2 Hz); ¹³C NMR (D₂O) 92.9, 71.3, 68.8,68.2, 59.8, 51.7; ³¹P NMR (D₂O) 2.80; HRMS: calcd for C₆H₁₃NO₈P258.0379, found m/z 258.0372 (M+H).

[0349] Disodium-(4-acetamido-4-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (92)).

[0350]Dibenzyl-(4-acetamido-2,3,6-tri-O-benzyl-4-deoxy-α-D-glucopyranosyl)phosphate (370 mg, 0.50 mmol) gave 120 mg (71%) of the desired product.[α]_(D)=109.2° (c=1, H₂O); ¹H NMR (D₂O) 5.44 (dd, 1H, J=3.3, 7.2 Hz),3.89 (m, 1H), 3.76 (m, 2H), 3.64 (dd, 1H, J 12.4, 1.2 Hz), 3.53 (m, 2H),1.99 (s, 3H); ¹³C NMR (D₂O) 173.8, 93.2, 71.4, 70.4, 69.8, 60.0, 50.6,21.3; ³¹P NMR (D₂O) 1.93; HRMS: calcd for C₈H₁₅NO₉P 300.0484, found m/z300.0499 (M+H).

[0351] Disodium-(6-amino-6-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (85)):

[0352] Dibenzyl-(6-azide-2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl)phosphate (360 mg, 0.49 mmol) gave 85 mg (57%) of the title compound. ¹HNMR (D₂O) 5.47 (dd, 1H, J=3.5, 6.8 Hz), 4.14 (dt, 1H, J=2.5, 12.6 Hz),3.78 (t, 1H, J=9.5 Hz), 3.55 (m, 2H), 3.33 (t, 1H, J=9.3 Hz), 3.07 (dd,1H, J=10.3, 12.9 Hz); ¹³C NMR (D₂O) 94.1, 73.4, 72.5, 72.4, 72.3, 68.6,41.0; ³¹P NMR (D₂O) 2.80; HRMS: calcd for C₆H₁₃NO₈P 258.0379, found m/z258.0388 (M+H).

[0353] Disodium-(6-acetamido-6-deoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (86)).

[0354]Dibenzyl-(6-acetamido-2,3,4-tri-O-benzyl-6-deoxy-α-D-glucopyranosyl)phosphate (340 mg, 0.45 mmol) gave 124 mg (79.4%) of the desiredproduct. [α]_(D)=60.5° (c=1, H₂O); ¹H NMR (D₂O) 5.39 (dd, 1H, J=3.2, 6.5Hz), 3.95 (t, 1H, J=7.1 Hz), 3.73 (t, 1H, J=9.4 Hz), 3.54 (m, 1H), 3.45(m, 1H), 3.34 (dd, 1H, J=6.7, 14.1 Hz), 3.25 (t, 1H, J=9.5 Hz), 1.99 (s,3H); ¹³C NMR (D₂O) 178.4, 97.4, 76.7, 75.9, 74.8, 73.8, 43.9, 25.6; ³¹PNMR (D₂O) 2.98; HRMS: calcd for C₈H₁₅NO₉P 300.0484, found m/z 300.0482(M+H)

[0355] Disodium-(4-amino-4,6-dideoxy-α-D-glucopyranosyl) Phosphate (FIG.3(b) (102))

[0356]Dibenzyl-(4-azide-3-O-benzoyl-2-O-benzyl-4,6-dideoxy-α-D-glucopyranosyl)phosphate (300 mg, 0.466 mmol) was dissolved in a mixture of 10 mL ofMeOH and 2 mL of toluene. To this solution was added 1.4 mL 1N NaOH and100 mg of 10% Pd/C and the reaction stirred overnight under hydrogenatmosphere. The catalyst was removed by filtration, the filtrateconcentrated to a volume of 4 mL, cooled to 0° C., and 0.7 mL 1N NaOHsolution was added in a dropwise fashion. The mixture was stirred for 3hr at 0° C., neutralized with 1N HOAc and the product purified via anionexchange as described in the general procedure above to give 86 mg (67%)of the substantially pure product. ¹H NMR (D₂O) 5.44 (dd, 1H, J=3.2, 6.7Hz), 4.24 (m, 1H), 3.88 (t, 1H, J=9.7 Hz), 3.56 (dd, 1H, J=1.3, 9.4 Hz),2.94 (t, 1H, J=10.3 Hz), 1.32 (d, 3H, J=6.2 Hz); ¹³C NMR (D₂O) 92.9,71.5, 68.3, 64.2, 56.6, 16.2; ³¹P NMR (D₂O) 2.16. HRMS: calcd forC₆H₁₃NO₇P 242.0429, found m/z 242.0441 (M+H)

[0357] E_(p)-Catalyzed Conversion.

[0358] A reaction containing 2.5 mM NTP, 5.0 mM sugar phosphate, 5.5 mMMgCl₂ and 10 U inorganic pyrophosphatase in a total volume of 50 μL 50mM potassium phosphate buffer, pH 7.5 at 37° C. was initiated by theaddition of 3.52 U E_(p) (1 U=the amount of protein needed to produce 1μmol TDP-α-D-glucose min⁻¹) The reaction was incubated with slowagitation for 30 min at 37° C., quenched with MeOH (50 μL), centrifuged(5 min, 14,000×g) and the supernatant was stored at −20° C. untilanalysis by HPLC. Samples (30 μL) were resolved on a Sphereclone 5u SAXcolumn (250×4.6 mm) fitted with a guard column (30×4.6 mm) using alinear gradient (50-200 mM potassium phosphate buffer, pH 5.0, 1.5 mLmin⁻¹, A₂₇₅ nm).

[0359] The following compounds were prepared, preferably according tothe methods described herein:

[0360] (109) Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z562.0837 (M+H).

[0361] (110) Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z564.0640 (M+H).

[0362] (111) Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z562.0848 (M+H).

[0363] (112) Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z564.0638 (M+H).

[0364] (113) Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z562.0835 (M+H).

[0365] (114) Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z564.0622 (M+H).

[0366] (115) Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₆H₂₆O₁₅N₃P₂ 562.0839; found m/z562.0842 (M+H).

[0367] (116) Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₅H₂₄O₁₆N₃P₂ 564.0632; found m/z564.0630 (M+H).

[0368] (117) Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z604.0953 (M+H).

[0369] (118) Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z606.0732 (M+H).

[0370] (119) Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z604.0940 (M+H).

[0371] (120) Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z606.0730 (M+H).

[0372] (121) Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z604.0947 (M+H).

[0373] (122) Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z606.0735 (M+H).

[0374] (123) Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₈H₂₈O₁₆N₃P₂ 604.0945; found m/z604.0951 (M+H).

[0375] (124) Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₇H₂₆O₁₇N₃P₂ 606.0737; found m/z606.0738 (M+H).

[0376] (125) Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₆H₂₆O₁₄N₃P₂ 546.0889; found m/z546.0895 (M+H).

[0377] (126) Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate). HRMS (FAB): calc for C₁₅H₂₄O₁₅N₃P₂ 548.0682; found m/z548.0673 (M+H).

[0378] Structure-Based Engineering of E_(p)

[0379] Expression, Purification and Mutagenesis of E_(p).

[0380] E_(p) may be modified in accordance with the present inventionaccording to the following method: E_(p) and E_(p) mutants are expressedand purified by methods known in the art. For seleno-methionine-labeledprotein, the expresion vector was transformed into the methionineauxotroph E. coli B834 and grown, preferably overnight at a temperatureof about 25° C. to about 35° C., preferably about 30° C. in the presenceof seleno-methionine. Seleno-methionine-labeled E_(p) is purified usingthe standard protocol but in the presence of DTT. All E_(p) mutant genecassettes are generated by a two-step PCR approach. Mutant genes aresubsequently characterized by dsDNA sequencing of both strands.

[0381] According to a preferred method, expression and purification andE_(p) and E_(p) mutants were accomplished as described in Jiang, J.,Biggins, J. B. & Thorson, J. S. A General Enzymatic Method for theSynthesis of Natural and “Unnatural” UDP- and TDP-Nucleotide Sugars. J.Am. Chem. Soc. 122, 6803-6804 (2000). For seleno-methionine-labeledprotein, the expresion vector was transformed into the methionineauxotroph E. coli B834 and grown overnight at 30° C. in the presence of50 mg L⁻¹ seleno-methionine. Seleno-methionine-labeled E_(p) waspurified using the standard protocol but in the presence of 5 mM DTT. Noadditional proteolysis or modifications during this process wereobserved by mass spectrometry. All E_(p) mutant gene cassettes weregenerated by a two-step PCR approach. Mutant genes were subsequentlycharacterized by dsDNA sequencing of both strands.

[0382] Crystallization. A general crystallization technique that may beused in accordance with the present invention, is as follows: PurifiedE_(p) is concentrated in a buffer, and crystallized in a hanging drop byvapor diffusion at approximately room temperature (20° C.). E_(p)-dTTPcrystals are obtained against reservoir containing TTP, 2.0 M ammoniumphosphate, 0.1 M Tris.HCl, pH 8.5, and 20 mM MgCl₂. Crystals grow withtwo monomers (half of the Ep tetramer) in the asymmetric unit. TheE_(p)-UDP-Glc crystals were obtained against a reservoir containing 2 mMUDP-Glc, 1.9 M ammonium sulfate, isopropanol.

[0383] According to an exemplary method, the purified E_(p) wasconcentrated to 20 mg mL⁻¹ in a buffer containing 10 mM KCl, 2 mM MgCl₂and 10 mM HEPES, pH 7.2, and crystallized in a hanging drop by vapordiffusion at room temperature (20° C.). The E_(p)-dTTP crystals wereobtained against reservoir containing 2 mM TTP, 2.0 M ammoniumphosphate, 0.1 M Tris.HCl, pH 8.5, and 20 mM MgCl₂. Crystals grow in thetetragonal space group P4₃2₁2 (a=b=120 Å, c=94 Å) with two monomers(half of the E_(p) tetramer) in the asymmetric unit. The E_(p)-UDP-Glccrystals were obtained against a reservoir containing 2 mM UDP-Glc, 1.9M ammonium sulfate, and 7.5% isopropanol. These crystals grow in theorthorhombic space group P2₁2₁2₁ (a=93 Å, b=112 Å, c=132 Å) with fourmonomers (one tetramer) in the asymmetric unit.

[0384] Data Collection and Structure Determination.

[0385] Data may be collected and structure determination made accordingto methods that would be known to those skilled in the art, including,for example, x-ray crystallography.

[0386] According to an exemplary embodiment, crystals were harvested andflash frozen in the cold stream of an X-Stream cooling system (Rigaku)in the mother liquor with added 20-25% glycerol as a cryoprotectant.Data was collected either in house using a Rigaku RAXIS-IV imaging platearea detector, or at the NSLS Brookhaven beamline X9B. Oscillationphotographs were integrated, scaled and merged using DENZO andSCALEPACK. (Otwinowski, Z. & Minor, W. Data Collection and Processing.,Sawyer, L., Isaacs, N. & Bailey, S. Ed. SERC Daresbury Laboratory:Warrington, UK. 556-562 (1993).) Subsequent calculations were performedwith the CCP4 program suite. (CCP4, The CCP4 suite: programs for X-raycrystallography. Acta Crystallogr. D, 50, 760-763 (1994).) TheE_(p)-UDP-Glc structure was determined using the single wavelengthanomalous diffraction phasing method. (Hendrickson, W. A., Determinationof Macromolecular Structures from Anomalous Diffraction of SynchrotronRadiation. Science, 254, 51-58 (1991).) Only the dataset collected atthe wavelength of the selenium absorption peak was processed. Peakwavelength anomalous data were input to the program SnB to identify thelocation of the Se atoms. Twenty peaks from the best solution wererefined using MLPHARE (CCP4) employing only the peak wavelengthanomalous differences in the resolution range 35 to 2A. Additional Sesites were located using anomalous-difference fourier maps. The finalround of MLPHARE consisted of 47 Se sites. Seven of these sitescorrespond to Se-methionines with dual sidechain conformation. Thephases calculated from MLPHARE had a figure of merit of 0.34 which wasimproved to 0.72 by density modification with the program DM (CCP4). Theresulting electron density map was clearly interpretable, indicatingalso the correct handedness of the Se substructure. The map was furtherimproved using free atom refinement and the automatic chain tracingprocedure of the wARP program. Out of the 1156 residues, the main chainof 1003 were automatically traced and very clear density could be seenfor the rest of the structure. The unambiguous tracing and sequenceassignment of the E_(p) tetramer was completed using the 0 program.(Jones, T. A., et al., Improved Methods for Building Protein Models inElectron Density Maps and the Location of Errors in these Models. ActaCrystallogr., A47, 110-119 (1991).) Refinement of the model byconventional least-squares algorithm was done with XPLOR. (Brünger, A.T., X-PLOR v. 3.1 Manual. New Haven: Yale University (1993).) The finalrefined E_(p) tetramer model at 2.0 A resolution had a free R-value(Brünger, A. T., Free R Value: A Novel Statistical Quantity forAssessing the Accuracy of Crystal Structures. Nature, 355, 472-475(1992)) of 22.3% and included 9938 non-hydrogen atoms in 1156well-ordered residues (1-289 in each monomer) and 762 water molecules.In our determination, electron density was lacking for only the 3C-terminal residues of each monomer. Restrained refinement oftemperature factors was monitored throughout by the free R-factorcriterion as set forth in Liu, H.-w. & Thorson, J. S. Pathways andMechanisms in the Biogenesis of Novel Deoxysugars by Bacteria. Ann. Rev.Microbiol. 48, 223-256 (1994) and Johnson, D. A. & Liu, H.-w. Mechanismsand Pathways from Recent Deoxysugar Biosynthesis Research. Curr. Opin.Chem. Biol. 2, 642-649 (1998)). Stereochemical analysis of the refinedmodel using PROCHECK (CCP4 suite) revealed main-chain and side-chainparameters better than, or within, the typical range of values forprotein structures determined at 2.0 Å resolution (overall G-factor,2.2). None of the E_(p) residues fell in the disallowed region of theRamachandran plot. (Ramachandran, G. N., Ramakrishnan, C. &Sasisekharan, V. Stereochemistry of Polypeptide Chain Configuration. J.Molec. Biol., 7, 95-99 (1963).) The E_(p)-dTTP structure was determinedusing the Molecular Replacement (MR) method, with our E_(p)-UDP-Glcstructure as a search model and the program XPLOR. The final refinedmodel (half of the E_(p) tetramer) at 2.1 Å resolution had a free Rvalue³⁵ of 23.5% and included 5017 non-hydrogen atoms in 578 welldefined in the electron density map amino acids (1-289 for eachmonomer), and 387 water molecules. The PROCHECK overall G-factor is 2.5,and none of the E_(p) residues fell in the disallowed region of theRamachandran plot.

[0387] Enzyme Assays and Determination of Steady State KineticParameters.

[0388] Assays for product formation and steady state kinetics wereaccomplished using conditions similar to those described. in Jiang, J.,Biggins, J. B. & Thorson, J. S. A General Enzymatic Method for theSynthesis of Natural and “Unnatural” UDP- and TDP-Nucleotide Sugars. J.Am. Chem. Soc. 122, 6803-6804 (2000). For the mutant pool assays, analiquot which contained an eqimolar ratio of each mutant (60 μg) wasutilized.

[0389] While the present invention is described with respect toparticular examples and preferred embodiments, it is understood that thepresent invention is not limited to these examples and embodiments. Thepresent invention as claimed therefore, includes variations from theparticular examples and preferred embodiments described herein, as willbe apparent to one of skill in the art.

What is claimed is:
 1. A nucleotidylyltransferase mutated at one or moreamino acids selected from the group consisting of V173, G147, W224,N112, G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146and Y177.
 2. The nucleotidylyltransferase of claim 1, wherein thenucleotidylyltransferase is E_(p).
 3. The nucleotidylyltransferase ofclaim 1, wherein the nucleotidylyltransferase is a purine or pyrimidinetriphosphate type nucleotidyltransferase set forth in FIG.
 19. 4. Apurine or pyrimidine triphosphate type nucleotidyltransferase set forthin FIG.
 19. 5. A nucleotidylyltransferase mutated such that it iscapable of having a different substrate specificity than a non-mutatednucleotidylyltransferase.
 6. The nucleotidylyltransferase of claim 5,having a substrate specificity for GTP, CTP, TTP, UTP, or ATP.
 7. Thenucleotidylyltransferase of claim 5, wherein thenucleotidylyltransferase is EP.
 8. The nucleotidylyltransferase of claim5, wherein the nucleotidyltransferase is mutated at one or more aminoacids selected from the group consisting of V173, G147, W224, N112,G175, D111, E162, T201, I200, E199, R195, L89, L89T, L109, Y146 andY177.
 9. A method comprising combining NTP and α-D-hexopyranosylphosphate in the presence of at least one mutatednucleotidylyltransferase.
 10. A nucleotide sugar produced by a methodcomprising combining NTP and α-D-hexopyranosyl phosphate in the presenceof at least one mutated nucleotidylyltransferase.
 11. The nucleotidesugar of claim 10, wherein the nucleotide sugar is selected from thegroup consisting of a TDP-sugar, a UDP-sugar, a GDP-sugar, a CDP-sugar,and an ADP-sugar.
 12. The nucleotide sugar of claim 10, selected fromthe group consisting of Thymidine 5′-(α-D-glucopyranosyl diphosphate)(58); Uridine 5′-(α-D-glucopyranosyl diphosphate) (59); Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60); Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61); Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62); Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63); Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64); Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65); Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66); Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67); Thymidine5′-(αD-mannopyranosyl diphosphate) (68); Uridine 5′-(α-D-mannopyranosyldiphosphate) (69); Thymidine 5′-(αD-galactopyranosyl diphosphate) (70);Uridine 5′-(αD-galactopyranosyl diphosphate) (71); Thymidine5′-(α-D-allopyranosyl diphosphate) (72); Uridine 5′-(αD-allopyranosyldiphosphate) (73); Thymidine 5′-(αD-altropyranosyl diphosphate) (74);Uridine 5′-(αD-altropyranosyl diphosphate) (75); Thymidine5′-(α-D-gulopyranosyl diphosphate) (76); Uridine 5′-(α-D-gulopyranosyldiphosphate) (77); Thymidine 5′-(αD-idopyranosyl diphosphate) (78);Uridine 5′-(α-D-idopyranosyl diphosphate) (79); Thymidine5′-(α-D-talopyranosyl diphosphate) (80); Uridine 5′-(α-D-talopyranosyldiphosphate) (81); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (109); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (110); Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (111); Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (112); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (113); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (114); Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (115); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (116); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (117); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (118); Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (119); Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (120); Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (121); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (122); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (123); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (124); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (125); Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (126);


13. A method comprising combining NTP and α-D-hexopyranosyl phosphateother than Glc1P in the presence of at least onenucleotidylyl-transferase.
 14. A nucleotide sugar produced by a methodcomprising combining NTP and α-D-hexopyranosyl phosphate other thanGlc1P in the presence of at least one nucleotidylyl-transferase.
 15. Amethod comprising combining α-D-hexopyranosyl phosphate and NTP otherthan TTP, in the presence of at least one nucleotidylyltransferase. 16.A nucleotide sugar produced by a method comprising combiningα-D-hexopyranosyl phosphate and NTP other than TTP, in the presence ofat least one nucleotidylyltransferase.
 17. A method of alteringnucleotidylyltransferase substrate specificity comprising mutating thenucleotidylyltransferase at one or more amino acids selected from thegroup consisting of V173, G147, W224, N112, G175, D111, E162, T201,I200, E199, R195, L89, L89T, L109, Y146 and Y177.
 18. The method ofclaim 17, wherein the nucleotidylyltransferase is EP.
 19. Anucleotidylyltransferase modified by the method of claim
 17. 20. Themodified nucleotidylyltransferase of claim 19, wherein thenucleotidylyltransferase is EP.
 21. A nucleotide sugar selected from thegroup consisting of Thymidine 5′-(α-D-glucopyranosyl diphosphate) (58);Uridine 5′-(α-D-glucopyranosyl diphosphate) (59); Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60); Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61); Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62); Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63); Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64); Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65); Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66); Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67); Thymidine5′-(α-D-mannopyranosyl diphosphate) (68); Uridine 5′-(α-D-mannopyranosyldiphosphate) (69); Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);Uridine 5′-(α-D-galactopyranosyl diphosphate) (71); Thymidine5′-(α-D-allopyranosyl diphosphate) (72); Uridine 5′-(α-D-allopyranosyldiphosphate) (73); Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);Uridine 5′-(α-D-altropyranosyl diphosphate) (75); Thymidine5′-(α-D-gulopyranosyl diphosphate) (76); Uridine 5′-(α-D-gulopyranosyldiphosphate) (77); Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);Uridine 5′-(α-D-idopyranosyl diphosphate) (79); Thymidine5′-(α-D-talopyranosyl diphosphate) (80); Uridine 5′-(α-D-talopyranosyldiphosphate) (81); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (109); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (110); Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (111); Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (112); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (113); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (114); Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (115); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (116); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (117); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (118); Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (119); Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (120); Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (121); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (122); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (123); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (124); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (125); Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (126);


22. A method of synthesizing nucleotide sugars comprising combing anucleotidylyltransferase, α-D-glucopyranosyl phosphate, NTP andignoranic pyrophosphatase, and incubating.
 23. The method of claim 22,comprising combining a nucleotidylyltransferase, α-D-glucopyranosylphosphate, Mg⁺², NTP and inorganic pyrophosphatase, and incubating at atemperature of from about 30° C. to about 45° C. for about 20 to about40 minutes.
 24. A method of synthesizing nucleotide sugars comprisingcombining a nucleotidylyltransferase, α-D-glucopyranosyl phosphate,Mg⁺², NTP and inorganic pyrophosphatase, and incubating; wherein thenucleotydylyltransferase is a natural nucleotidylyltransferase.
 25. Amethod of synthesizing nucleotide sugars comprising combining anucleotidylyltransferase, α-D-glucopyranosyl phosphate, Mg⁺², NTP andinorganic pyrophosphatase, and incubating; wherein thenucleotidylyltransferase is a mutated nucleotidylyltransferase.
 26. Themethod of claim 9, wherein the mutated nucleotidylyltransferase is anucleotidylyltransferase mutated at one or more amino acids selectedfrom the group consisting of V173, G147, W224, N112, G175, D111, E162,T201, I200, E199, R195, L89, L89T, L109, Y146 and Y177.
 27. The methodof claim 25, wherein the mutated nucleotidylyltransferase is anucleotidylyltransferase mutated at one or more amino acids selectedfrom the group consisting of V173, G147, W224, N112, G175, D111, E162,T201, I200, E199, R195, L89, L89T, L109, Y146 and Y177.
 28. The methodof claim 26, wherein the modified nucleotidylyltransferase is EP. 29.Nucleotide sugars sythesized by the method of claim
 22. 30. Nucleotidesugars sythesized by the method of claim
 24. 31. Nucleotide sugarssythesized by the method of claim
 25. 32. Nucleotide sugars sythesizedby the method of claim 22, selected from the group consisting ofThymidine 5′-(α-D-glucopyranosyl diphosphate) (58); Uridine5′-(α-D-glucopyranosyl diphosphate) (59); Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60); Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61); Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62); Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63); Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64); Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65); Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66); Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67); Thymidine5′-(α-D-mannopyranosyl diphosphate) (68); Uridine 5′-(α-D-mannopyranosyldiphosphate) (69); Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);Uridine 5′-(α-D-galactopyranosyl diphosphate) (71); Thymidine5′-(α-D-allopyranosyl diphosphate) (72); Uridine 5′-(αD-allopyranosyldiphosphate) (73); Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);Uridine 5′-(α-D-altropyranosyl diphosphate) (75); Thymidine5′-(α-D-gulopyranosyl diphosphate) (76); Uridine 5′-(α-D-gulopyranosyldiphosphate) (77); Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);Uridine 5′-(α-D-idopyranosyl diphosphate) (79); Thymidine5′-(α-D-talopyranosyl diphosphate) (80); and Uridine5′-(α-D-talopyranosyl diphosphate) (81).
 33. Nucleotide sugarssythesized by the method of claim 9, selected from the group consistingof Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (109);Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (110);Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (111);Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (112);Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (113);Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (114);Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (115);Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (116);Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (117);Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (118);Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (119);Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (120);Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (121);Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (122);Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (123);Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (124);Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (125);and Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate)(126).
 34. Nucleotide sugars sythesized by the method of claim 9,selected from the group consisting of


35. A α-D-hexopyranosyl phosphate comprising at least one phosphateselected from the group consisting of deoxy-α-D-glucopyranosyl,aminodeoxy-α-D-hexapyranosyl, and acetamidodeoxy-α-D-hexopyranosylphosphates.
 36. A method comprising combining a hexopyranosyl phosphateaccording to claim 35 and NTP in the presence of anucleotidylyltransferase.
 37. The method of claim 36, wherein thenucleotidylyltransferase is mutated.
 38. A nucleotide sugar produced bythe method of claim
 36. 39. A nucleotide sugar library comprising two ormore nuceleotide sugars produced by combining α-D-hexopyranosylphosphate and NTP in the presence of at least one mutatednucleotidylyltransferase.
 40. A nucleotide sugar library comprising twoor more nucleotide sugars produced by combining α-D-hexopyranosylphosphate other than Glc1P and NTP in the presence of at least onenucleotidylyltransferase.
 41. A nucleotide sugar library comprising twoor more nuceleotide sugars produced by combining α-D-hexopyranosylphosphate and NTP other than TTP in the presence of at least onenucleotidylyltransferase.
 42. A nucleotide sugar library comprising twoor more nuceleotide sugars selected from the group consisting ofThymidine 5′-(α-D-glucopyranosyl diphosphate) (58); Uridine5′-(α-D-glucopyranosyl diphosphate) (59); Thymidine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60); Uridine5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61); Thymidine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62); Uridine5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63); Thymidine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64); Uridine5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65); Thymidine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66); Uridine5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67); Thymidine5′-(α-D-mannopyranosyl diphosphate) (68); Uridine 5′-(α-D-mannopyranosyldiphosphate) (69); Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);Uridine 5′-(α-D-galactopyranosyl diphosphate) (71); Thymidine5′-(α-D-allopyranosyl diphosphate) (72); Uridine 5′-(α-D-allopyranosyldiphosphate) (73); Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);Uridine 5′-(α-D-altropyranosyl diphosphate) (75); Thymidine5′-(α-D-gulopyranosyl diphosphate) (76); Uridine 5′-(α-D-gulopyranosyldiphosphate) (77); Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);Uridine 5′-(α-D-idopyranosyl diphosphate) (79); Thymidine5′-(α-D-talopyranosyl diphosphate) (80); Uridine 5′-(α-D-talopyranosyldiphosphate) (81); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (109); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyldiphosphate) (110); Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (111); Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyldiphosphate) (112); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (113); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyldiphosphate) (114); Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (115); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyldiphosphate) (116); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (117); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyldiphosphate) (118); Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (119); Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyldiphosphate) (120); Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (121); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyldiphosphate) (122); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (123); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyldiphosphate) (124); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (125); Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyldiphosphate) (126);


43. A method comprising incubating a glycotransferase with one or moreof the sugars of the nucleotide sugar library of claim 39, and amolecule capable of being glycosylated.
 44. A method comprisingincubating a glycotransferase with one or more of the sugars of thenucleotide sugar library of claim 40, and a molecule capable of beingglycosylated.
 45. A method comprising incubating a glycotransferase withone or more of the sugars of the nucleotide sugar library of claim 41,and a molecule capable of being glycosylated.
 46. A method comprisingincubating a glycotransferase with one or more of the sugars of thenucleotide sugar library of claim 42, and a molecule capable of beingglycosylated.
 47. A method of making α-D-hexopyranosyl phosphatescomprising anomerically activating an ethyl 1-thio-β-D-pyranoside toform a compound having the formula

wherein R² is OCH₃, OBz, or OH, R³ is OH, OAc, or OBz, R⁴ is H, OH, or ahalogen atom, and R⁵, R⁶, R⁷, R⁸, and R⁹ are each OBz, and three or moreof R³, R⁵, R⁶, R⁷, R⁸, and R⁹ are OBz substituents; deprotecting the OBzsubstituents to convert at least one such substituent to a OBnsubstituent; phosphorylating to form a compound of the formula

wherein R¹ is OCH₃, OBz, OAc or OH, R is OCH₃, OBz, OAc or OH, R³ is OH,OAc, or OBz, R⁴ is H, OH, OBz, OAc or a halogen atom, R⁵, R⁶, R⁷, R⁸,and R⁹ are each OBz or OAc, and R¹⁰ is OH, or OBn, wherein at least fourof R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently OBn or OBzsubstituents; and deprotecting to convert any OBn substituents to OHsubstituents.
 48. An α-D-hexopyranosyl phosphate prepared according tothe method of claim
 47. 49. The α-D-hexopyranosyl phosphate of claim 48,whierein the α-D-hexopyranosyl phosphate is a glycosyl phosphate. 50.The α-D-hexopyranosyl phosphate of claim 48, selected from the groupconsisting of deoxy-α-D-glucopyranosyl, aminodeoxy-α-D-hexopyranosyl andacetamidodeoxy-α-D-hexopyranosyl phosphates.
 51. A method comprisingproviding isolated E_(p) having the formula

wherein R¹ is OCH₃, OBz, OAc or OH, R² is OCH₃, OBz, OAc or OH, R³ isOH, OAc, or OBz, R⁴ is H, OH, OBz, OAc or a halogen atom, R⁵, R⁶, R⁷,R⁸, and R⁹ are each OBz or OAc, and R¹⁰ is OH, or OBn, wherein at leastfour of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are independently OH or OBzsubstituents.
 52. A method of making aminodeoxy-α-D-glucopyranosylphosphates comprising converting an intermediate selected from the groupconsisting of ethyl6-azide-2,3,4-tri-O-benzyl-6-deoxy-1-thio-β-D-glucopyranoside (89),ethyl 4-azide-2,3,6 tri-O-benzyl-4-deoxy-1-thio-β-D-glucopyranoside(94), and ethyl3-azide-2,4,6-tri-O-benzyl-3-deoxy-1-thio-β-D-glucopyranoside (100) to acorresponding amide.
 53. A method of making α-D-hexopyranosyl phosphatescomprising phosphorylating a phosphate selected from the groupconsisting of Dibenzyl-(2,3,4,6-tetra-O-benzyol-α-D-altropyranosyl)phosphate, Dibenzyl-(2,3,4,6-tetra-O-benzyl-α-D-idopyranoxyl)phosphate,and Dibenzyl-(2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl) phosphate via aglycosyl halide; and deprotecting to form a glycosyl phosphate selectedfrom the group conssiting of Disodium α-D-altropyranosyl phosphate,Disodium α-Didopyranosyl phosphate and Disodium α-D-talopyranosylphosphate.
 54. An α-D-hexopyranosyl phosphate prepared according to themethod of claim 53.